Flow cell with one or more barrier features

ABSTRACT

An apparatus includes a flow cell body, a plurality of electrodes, an imaging assembly, and one or more barrier features. The flow cell body defines one or more flow channels and a plurality of wells defined as recesses in the floor of each flow channel. Each well is fluidically coupled with the corresponding flow channel. The flow cell body further defines interstitial surfaces between adjacent wells. Each well defines a corresponding depth. Each electrode is positioned in a corresponding well of the plurality of wells. The electrodes are to effect writing of polynucleotides in the wells. The imaging assembly is to capture images of polynucleotides written in the wells. The one or more barrier features are positioned in the wells, between the wells, or above the wells. The one or more barrier features contain reactions in each well, reduce diffusion between the wells, or reduce optical cross-talk between the wells.

RELATED APPLICATIONS

This application is a national stage entry of International PatentApplication No. PCT/US2020/034518, entitled “Flow Cell with One or MoreBarrier Features,” filed on May 26, 2020, which claims priority to U.S.Provisional Patent App. No. 62/855,654, entitled “Flow Cell with One orMore Barrier Features In or Between Wells,” filed on May 31, 2019, whichis incorporated by reference herein in its entirety. InternationalPatent Application No. PCT/US2020/034518 also claims priority to U.S.Provisional Patent App. No. 62/855,662, entitled “Flow Cell with BarrierFeatures to Prevent Optical Cross-Talk Between Wells,” filed on May 31,2019, which is incorporated by reference herein in its entirety.

BACKGROUND

Computer systems have used various different mechanisms to store data,including magnetic storage, optical storage, and solid-state storage.Such forms of data storage may present drawbacks in the form ofread-write speed, duration of data retention, power usage, or datadensity.

Just as naturally occurring DNA may be read, machine-written DNA mayalso be read. Pre-existing DNA reading techniques may include anarray-based, cyclic sequencing assay (e.g., sequencing-by-synthesis(SBS)), where a dense array of DNA features (e.g., template nucleicacids) are sequenced through iterative cycles of enzymatic manipulation.After each cycle, an image may be captured and subsequently analyzedwith other images to determine a sequence of the machine-written DNAfeatures. In another biochemical assay, an unknown analyte having anidentifiable label (e.g., fluorescent label) may be exposed to an arrayof known probes that have predetermined addresses within the array.Observing chemical reactions that occur between the probes and theunknown analyte may help identify or reveal properties of the analyte.

SUMMARY

Described herein are devices, systems, and methods for containingreactions within wells of a DNA storage device and to prevent orotherwise reduce diffusion between wells of the DNA storage device.

An implementation relates to an apparatus comprising a flow cell body.In some such implementations, the flow call body may define one or moreflow channels and a plurality of wells, each channel of the one or moreflow channels to receive a fluid. In some such implementations, eachwell of the plurality of wells may be fluidically coupled with thecorresponding flow channel of the one or more flow channels, and eachwell of the plurality of wells may define a corresponding depth. In somesuch implementations, the apparatus may also comprise a plurality ofelectrodes. In some such implementations, each electrode of theplurality of electrodes may be positioned in a corresponding well of theplurality of wells, the plurality of electrodes to effect writing ofpolynucleotides in the corresponding wells of the plurality of wells. Insome such implementations, the apparatus may also comprise an imagingassembly to capture images indicative of one or more nucleotides in apolynucleotide. In some such implementations, the apparatus may compriseone or more barrier features positioned in or between the plurality ofwells, the barrier features to contain reactions in each well of theplurality of wells or reduce diffusion between the plurality of wells.

Variations on any one or more of the above implementations exist,wherein each electrode of the plurality of electrodes may define anaperture.

Variations on any one or more of the above implementations exist,wherein the imaging assembly may include at least one image sensor toreceive light through the aperture of each electrode of the plurality ofelectrodes.

Variations on any one or more of the above implementations exist,wherein each electrode of the plurality of electrodes may be annularlyshaped.

Variations on any one or more of the above implementations exist,wherein each electrode of the plurality of electrodes may comprise aplurality of electrode segments arranged in quadrants, the aperturebeing defined at a central region of the arrangement of quadrants.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise an integrated circuitpositioned under the flow cell body, the integrated circuit to drive theelectrodes to thereby effect writing of polynucleotides in thecorresponding wells of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the integrated circuit may be further in communication with theimaging assembly.

Variations on any one or more of the above implementations exist,wherein the integrated circuit may comprise a complementarymetal-oxide-semiconductor (CMOS) chip.

Variations on any one or more of the above implementations exist,wherein the plurality of wells may be formed as a plurality of discreterecesses arranged in a pattern along a base surface of the correspondingflow channel of the one or more flow channels.

Variations on any one or more of the above implementations exist,wherein each well of the plurality of wells may be defined by at leastone sidewall and a floor.

Variations on any one or more of the above implementations exist,wherein each electrode of the plurality of electrodes may be positionedon the floor of the corresponding well of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein each electrode of the plurality of electrodes may be positionedon a sidewall of the at least one sidewall of the corresponding well ofthe plurality of wells.

Variations on any one or more of the above implementations exist,wherein the floor of each well of the plurality of wells may furtherdefine an opening, the opening to provide a path for fluid communicationbetween the corresponding well of the plurality of wells and a fluidsource.

Variations on any one or more of the above implementations exist,wherein the barrier features may comprise a plurality of valves, eachvalve of the plurality of valves being positioned in the opening of eachcorresponding well of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein each valve of the plurality of valves may comprise a hydrogelmaterial.

Variations on any one or more of the above implementations exist,wherein the hydrogel material to transition between an expanded stateand a contracted state, the hydrogel material to effectively close theaperture of the well of the plurality of wells in the expanded state,the hydrogel material to effectively open the aperture of the well ofthe plurality wells in the contracted state.

Variations on any one or more of the above implementations exist,wherein each valve of the plurality of valves may comprise a hydrogelring defining an opening aligned with the aperture of the correspondingwell of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein each valve of the plurality of valves may comprise anelectroactive polymer material.

Variations on any one or more of the above implementations exist,wherein each valve of the plurality of valves to transition between anopen state and a closed state in response to changes in pH. In some suchimplementations, the apparatus may further comprise a pH driving featureto thereby selectively transition the valve of the plurality of valvesbetween the open state and the closed state.

Variations on any one or more of the above implementations exist,wherein each valve of the plurality of valves to transition between anopen state and a closed state in response to changes in temperature. Insome such implementations, the apparatus may further comprise atemperature driving feature to selectively vary a temperature valueassociated with each valve of the plurality of valves to therebyselectively transition the valve of the plurality of valves between theopen state and the closed state.

Variations on any one or more of the above implementations exist,wherein each valve of the plurality of valves may comprise a heatswellable polymer.

Variations on any one or more of the above implementations exist,wherein each valve of the plurality of valves being biased toward aclosed state, each valve of the plurality of valves to open in responseto fluid pressure against the valve of the plurality of valves exceedinga threshold.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise a control module to controlopening and closing of each valve of the plurality of valves.

Variations on any one or more of the above implementations exist,wherein the control module to selectively activate the plurality ofvalves to allow deblocking agent to pass through the apertures of theplurality of wells.

Variations on any one or more of the above implementations exist,wherein the one or more barrier features may include a flow gradientgenerator, the flow gradient generator to provide a fluid flow profilethat varies across the depth of each well of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the flow gradient generator to provide a reduced flow of fluidor fluid pressure at a bottom region of each well of the plurality ofwells, with an increased flow of fluid or fluid pressure at a top regionof each well of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the flow gradient generator may comprise a pump.

Variations on any one or more of the above implementations exist,wherein the flow gradient generator may comprise a bubble generator.

Variations on any one or more of the above implementations exist,wherein the one or more barrier features may include a temperaturegradient generator, the temperature gradient generator to provide atemperature profile that varies across the depth of each well of theplurality of wells.

Variations on any one or more of the above implementations exist,wherein the temperature gradient generator to provide a relativelyhigher temperature in a bottom region of each well of the plurality ofwells and a relatively lower temperature in a top region of each well ofthe plurality of wells.

Variations on any one or more of the above implementations exist,wherein the one or more barrier features may include a temperaturegradient generator, the temperature gradient generator to provide atemperature profile that varies between the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the temperature gradient generator to provide a relativelyhigher temperature within each well of the plurality of wells and arelatively lower temperature in spaces between the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the one or more barrier features may comprise a plurality ofhydrophobic partitions positioned along a base surface of the flow cellbody between adjacent wells of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the plurality of hydrophobic partitions may extend from the basesurface of the flow cell body toward an upper surface of the flow cellbody.

Variations on any one or more of the above implementations exist,wherein the plurality of hydrophobic partitions to transition frompositions along the base surface of the flow cell body between adjacentwells of the plurality of wells to positions over each correspondingwell of the plurality of wells, such that the plurality of hydrophobicpartitions are to transition from being barriers between the pluralityof wells to being caps over the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the plurality of hydrophobic partitions to move in response toan applied voltage.

Variations on any one or more of the above implementations exist,wherein each hydrophobic partition of the plurality of hydrophobicpartitions to cover a two more wells of the plurality of wellssimultaneously.

Variations on any one or more of the above implementations exist,wherein each hydrophobic partition of the plurality of hydrophobicpartitions to cover only a single well of the plurality of wells, suchthat each of the plurality of wells has a respective hydrophobicpartition of the plurality of hydrophobic partitions.

Variations on any one or more of the above implementations exist,wherein each hydrophobic partition of the plurality of hydrophobicpartitions may comprise a volume of a fluid.

Variations on any one or more of the above implementations exist,wherein each hydrophobic partition of the plurality of hydrophobicpartitions may comprise a volume of an oil.

Variations on any one or more of the above implementations exist,wherein the one or more barrier features may comprise a pH drivingfeature to selectively adjust pH values associated with each well of theplurality of wells to thereby provide variation in pH values amongadjacent wells of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein pH driving feature may comprise a bubble generator.

Variations on any one or more of the above implementations exist,wherein the pH driving feature may comprise electrodes.

Variations on any one or more of the above implementations exist,wherein the plurality of electrodes to effect writing of polynucleotidesin the form of DNA strands in the corresponding wells of the pluralityof wells.

Variations on any one or more of the above implementations exist,wherein the plurality of electrodes may include electrodes positioned ina bottom region of each well of the plurality of wells, the electrodespositioned in the bottom region of each well of the plurality of wellsto drive a redox reaction within the corresponding well of the pluralityof wells.

Variations on any one or more of the above implementations exist,wherein each electrode of the electrodes positioned in the bottom regionof each well of the plurality of wells may comprise a copper element.

Variations on any one or more of the above implementations exist,wherein the plurality of electrodes may further include electrodespositioned on surfaces of interstitial spaces between wells of theplurality of wells, the electrodes on surfaces of interstitial spacesbetween wells of the plurality of wells providing the one or morebarrier features.

Variations on any one or more of the above implementations exist,wherein the electrodes positioned on surfaces of interstitial spacesbetween wells of the plurality of wells to provide a reverse currentthat sharpens boundaries defined by the electrodes positioned in thebottom region of each well of the plurality of wells.

Another implementation relates to an apparatus comprising a bodydefining an upper flow channel a plurality of wells, the upper flowchannel to receive a flow of fluid containing nucleotides. In some suchimplementations, each well of the plurality of wells may be fluidicallycoupled with the corresponding flow channel, and each well of theplurality of wells may have a floor with an aperture. In some suchimplementations, the apparatus may comprise a lower channel positionedunder the floor of each well of the plurality of wells, the lowerchannel to receive fluid containing a deblocking agent. In some suchimplementations, the apparatus may comprise a plurality of valves. Insome such implementations, each valve of the plurality of valves totransition between an open and closed state, each valve of the pluralityof valves to permit fluid communication between the lower channel andthe corresponding well of the plurality of wells in the open state, eachvalve of the plurality of valves to reduce fluid communication betweenthe lower channel and the corresponding well of the plurality of wellsin the closed state. In some such implementations, the apparatus maycomprise a plurality of electrodes. In some such implementations, eachelectrode of the plurality of electrodes may be positioned in acorresponding well of the plurality of wells, the plurality ofelectrodes to effect writing of polynucleotides in the correspondingwells of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise an imaging assembly tocapture images indicative of one or more nucleotides in apolynucleotide.

Variations on any one or more of the above implementations exist,wherein each valve of the plurality of valves may comprise a hydrogelmaterial.

Variations on any one or more of the above implementations exist,wherein each valve of the plurality of valves may comprise anelectroactive polymer.

Variations on any one or more of the above implementations exist,wherein each valve of the plurality of valves may comprise a heatswellable polymer.

Variations on any one or more of the above implementations exist,wherein each valve of the plurality of valves may be biased toward aclosed state, each valve of the plurality of valves to open in responseto fluid pressure against the valve exceeding a threshold.

Yet another implementation relates to an apparatus comprising a flowcell body. In some such implementations, the flow cell body may defineone or more flow channels and a plurality of wells, each flow channel ofthe one or more flow channels to receive a flow of fluid. In some suchimplementations, each well of the plurality of wells may be fluidicallycoupled with the corresponding flow channel of the one or more flowchannels, and each well of the plurality of wells may define acorresponding depth. In some such implementations, the apparatus maycomprise a plurality of electrodes. In some such implementations, eachelectrode of the plurality of electrodes may be positioned in acorresponding well of the plurality of wells, the plurality ofelectrodes to effect writing of polynucleotides in the correspondingwells of the plurality of wells. In some such implementations, theapparatus may comprise a flow gradient generator, the flow gradientgenerator to provide a fluid flow profile that varies across the depthof each well of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise an imaging assembly tocapture images indicative of one or more nucleotides in apolynucleotide.

Variations on any one or more of the above implementations exist,wherein the flow gradient generator to provide a reduced flow of fluidor fluid pressure at a bottom region of each well of the plurality ofwells, with an increased flow of fluid or fluid pressure at a top regionof each well of the plurality of wells.

Yet another implementation relates to an apparatus comprising a flowcell body. In some such implementations, the flow cell body may defineone or more flow channels and a plurality of wells, each flow channel ofthe one or more flow channels to receive a flow of fluid. In some suchimplementations, each well of the plurality of wells may be fluidicallycoupled with the corresponding flow channel of the one or more flowchannels, and each well of the corresponding wells defining acorresponding depth. In some such implementations, the apparatus maycomprise a plurality of electrodes. In some such implementations, eachelectrode from the plurality of electrodes may be positioned in acorresponding well of the plurality of wells, the plurality ofelectrodes to effect writing of polynucleotides in the correspondingwells of the plurality of wells. In some such implementations, theapparatus may comprise a temperature gradient generator, the temperaturegradient generator to provide a temperature profile that varies acrossthe depth of each well of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise an imaging assembly tocapture images indicative of one or more nucleotides in apolynucleotide.

Variations on any one or more of the above implementations exist,wherein the temperature gradient generator to provide a relativelyhigher temperature in a bottom region of each well of the plurality ofwells and a relatively lower temperature in a top region of each well ofthe plurality of wells.

Yet another implementation relates to an apparatus comprising a flowcell body defining one or more channels and a plurality of wells, eachflow channel of the one or more flow channels to receive a flow offluid. In some such implementations, each well of the plurality of wellsmay be fluidically coupled with the corresponding flow channel of theone or more flow channels, each well of the plurality of wells defininga corresponding depth. In some such implementations, the apparatus maycomprise a plurality of electrodes. In some such implementations, eachelectrode of the plurality of electrodes may be positioned in acorresponding well of the plurality of wells, the plurality ofelectrodes to effect writing of polynucleotides in the correspondingwells of the plurality of wells. In some such implementations, theapparatus may comprise a temperature gradient generator, the temperaturegradient generator to provide a temperature profile that varies betweenthe plurality of wells.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise an imaging assembly tocapture images indicative of one or more nucleotides in apolynucleotide.

Variations on any one or more of the above implementations exist,wherein the temperature gradient generator to provide a relativelyhigher temperature within each well of the plurality of wells, and arelatively lower temperature in spaces between the plurality of wells.

Yet another implementation relates to an apparatus comprising a flowcell body defining one or more flow channels, an upper surface, a basesurface, and a plurality of wells formed in the base surface, each flowchannel of the one or more flow channels to receive a flow of fluid,each well of the plurality of wells being fluidically coupled with thecorresponding flow channel of the one or more flow channels. In somesuch implementations, the apparatus may comprise a plurality ofelectrodes. In some such implementations, each electrode of theplurality of electrodes may be positioned in a corresponding well of theplurality of wells, the plurality of electrodes to effect writing ofpolynucleotides in the corresponding wells of the plurality of wells. Insome such implementations, the apparatus may comprise a plurality ofhydrophobic partitions positioned along the base surface of the flowcell body between adjacent wells of the plurality of wells. In some suchimplementations, the plurality of hydrophobic partitions may extend fromthe base surface of the flow cell body toward an upper surface of theflow cell body.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise an imaging assembly tocapture images indicative of one or more nucleotides in apolynucleotide.

Variations on any one or more of the above implementations exist,wherein the plurality of hydrophobic partitions to move from positionsalong the floor of the flow cell body between adjacent wells of theplurality of wells to positions over each corresponding well of theplurality of wells, such that the plurality of hydrophobic partitionsare to transition from being barriers between the plurality of wells tobeing caps over the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the plurality of hydrophobic partitions to move in response toan applied voltage.

Variations on any one or more of the above implementations exist,wherein each hydrophobic partition of the plurality of hydrophobicpartitions may comprise a volume of fluid.

Yet another implementation relates to an apparatus comprising a flowcell body defining one or more channels, an upper surface, a basesurface, and a plurality of wells formed in the base surface, each flowchannel of the one or more flow channels to receive a flow of fluid. Insome such implementations, each well of the plurality of wells may befluidically coupled with the one or more flow channels. In some suchimplementations, the apparatus may comprise a plurality of electrodes.In some such implementations, each electrode of the plurality ofelectrodes may be positioned in a corresponding well of the plurality ofwells, the plurality of electrodes to effect writing of polynucleotidesin the corresponding wells of the plurality of wells. In some suchimplementations, the apparatus may comprise a pH driving feature toselectively adjust pH values associated with each well of the pluralityof wells to thereby provide variation in pH values among adjacent wellsof the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise an imaging assembly tocapture images of polynucleotides written in the wells of the pluralityof wells.

Variations on any one or more of the above implementations exist,wherein the pH driving feature may comprise a device selected from thegroup consisting of a bubble generator and a set of electrodes.

An implementation relates to an apparatus comprising a flow cell bodydefining one or more flow channels, each flow channel of the one or moreflow channels to receive a flow of fluid. In such implementations, atleast one of the one or more flow channels may have a floor and aplurality of wells defined as recesses in the floor, each well of theplurality of wells being fluidically coupled with the corresponding flowchannel, the flow cell body further defining interstitial spaces betweenadjacent wells of the plurality of wells. In some such implementations,the apparatus may comprise a plurality of electrode assemblies. In somesuch implementations, each electrode assembly of the plurality ofelectrode assemblies may be positioned in a corresponding well of theplurality of wells, the plurality of electrode assemblies to generatemachine-written polynucleotides in the corresponding wells of theplurality of wells. In some such implementations, the apparatus maycomprise one or more barrier features positioned above the plurality ofwells or in the plurality of wells, the one or more barrier features toreduce optical cross-talk between the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise an imaging assembly tocapture images indicative of one or more nucleotides in machine-writtenpolynucleotides written in the wells of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the one or more barrier features may comprise an electricallyactivated polarizer.

Variations on any one or more of the above implementations exist,wherein each of the polarizers may be positioned in an upper region of acorresponding well of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein each of the polarizers may define an opening to permit fluid toflow from the at least one of the one or more flow channels into thecorresponding well of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise a light source, the lightsource to project light to two or more wells of the plurality of wellsthat are positioned to simultaneously receive the light projected fromthe light source.

Variations on any one or more of the above implementations exist,wherein the polarizers to selectively permit or restrict passage oflight from the light source to the two or more wells that are positionedto simultaneously receive the light projected from the light source.

Variations on any one or more of the above implementations exist,wherein the one or more barrier features may comprise photoguides.

Variations on any one or more of the above implementations exist,wherein the plurality of electrode assemblies to generatemachine-written polynucleotides in the form of DNA strands.

Another implementation relates to a method comprising flowing fluidthrough a flow cell. In some such implementations, the flow may containnucleotides, and the flow cell may comprise a flow cell body definingone or more flow channels, each flow channel of the one or more flowchannels to receive a flow of fluid. In some such implementations, atleast one of the one or more flow channels may have a floor, a pluralityof wells defined as recesses in the floor, and a plurality of barriersabove the wells of the plurality of wells or in the wells of theplurality of wells. In some such implementations, each well of theplurality of wells may be fluidically coupled with the correspondingflow channel of the one or more flow channels, and the floor of the flowchannel of the one or more flow channels may interstitial spaces betweenadjacent wells of the plurality of wells. In some such implementations,the method may comprise activating electrode assemblies at bottomregions of each well of the plurality of wells to generatemachine-written polynucleotides within each well of the plurality ofwells, the machine-written polynucleotides representing stored data. Insome such implementations, the method may comprise activating theplurality of barrier features to reduce optical cross-talk betweenadjacent wells of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein activating the electrode assemblies at the bottom region of eachwell of the plurality of wells may comprise generating a positivecurrent and activating the plurality of barrier features may comprisegenerating a negative current.

Variations on any one or more of the above implementations exist,wherein activating the plurality of barrier features may comprise addinga charge tag to at least some of the nucleotides.

Variations on any one or more of the above implementations exist,wherein each charge tag may be positively charged.

Variations on any one or more of the above implementations exist,wherein each charge tag may impart a net positive charge to thecorresponding nucleotide of the nucleotides.

Variations on any one or more of the above implementations exist,wherein each charge tag may be negatively charged.

Variations on any one or more of the above implementations exist,wherein each charge tag may be attached to one or more regions of thecorresponding nucleotide of the nucleotides selected from a ribose, aphosphate group, or a base.

Variations on any one or more of the above implementations exist,wherein each charge tag may be cleaved before a ligation event.

Variations on any one or more of the above implementations exist,wherein each charge tag may be cleaved after a ligation event.

Yet another implementation relates to a method comprising flowing afluid through a flow cell, the fluid containing nucleotides. In somesuch implementations, the flow cell may comprise a flow cell bodydefining one or more flow channels, each flow channel of the one or moreflow channels to receive a flow of fluid. In some such implementations,each flow channel of the one or more flow channels may have a floor, anda plurality of wells defined as recesses in the floor. In some suchimplementations, each well of the plurality of wells may be fluidicallycoupled with the corresponding flow channel of the one or more flowchannels, the floor of the flow channel of the one or more flow channelsdefining interstitial surfaces between adjacent wells of the pluralityof wells. In some such implementations, each flow channel of the one ormore flow channels may have a plurality of barrier features positionedabove the plurality of wells or in the plurality of wells, the pluralityof barrier features to reduce optical cross-talk between the pluralityof wells. In some such implementations, the method may comprisedetermining a first subset of the plurality of wells that are unused,and that are not adjacent to a used well of the plurality of wells. Insome such implementations, the method may comprise activating electrodeassemblies at bottom regions of each well of the first subset togenerate machine-written polynucleotides within each well of the firstsubset, the machine-written polynucleotides within each well of thefirst subset representing stored data, while leaving the plurality ofbarrier features deactivated. In some such implementations, the methodmay comprise determining a second subset of the plurality of wells thatare unused, but that are adjacent to a used well of the plurality ofwells. In some such implementations, the method may comprise activatingelectrode assemblies at bottom regions of each well of the second subsetto generate machine-written polynucleotides within each well of thesecond subset, the machine-written polynucleotides the machine-writtenpolynucleotides within each well of the second subset representingstored data, after activating the barrier features to reduce opticalcross-talk between adjacent wells of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the method may comprise receiving a request for stored data. Insome such implementations, the method may comprise determining a set oftarget wells from the plurality of wells in which the stored data isrepresented by machine-written polynucleotides. In some suchimplementations, the method may comprise determining whether each wellof the set of target wells is adjacent to any used wells of theplurality of wells. In some such implementations, the method maycomprise, where a target well of the set of target wells is adjacent toany used well, activating an integrated circuit to detect fluorescenceemitted from a fluorophore of a machine-written polynucleotide and readthe stored data from the target well of the set of target wells that isadjacent to a used well, after activating the barrier features to reduceoptical cross-talk with the adjacent wells of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the method may comprise, where the target well is adjacent toany used well, performing a cross-talk quality control check on the readstored data. In some such implementations, the method may comprise,where the cross-talk quality control check verifies that that readstored data is not corrupted, providing the read stored data to arequesting device.

Variations on any one or more of the above implementations exist,wherein the method may comprise receiving a request for stored data. Insome such implementations, the method may comprise determining a set oftarget wells from the plurality of wells in which the stored data isrepresented by machine-written polynucleotides. In some suchimplementations, the method may comprise determining whether each wellof the set of target wells is adjacent to any used wells of theplurality of wells. In some such implementations, the method maycomprise, where a target well of the set of target wells is not adjacentto any used well, activating an integrated circuit to detectfluorescence of emitted from a fluorophore of a machine-writtenpolynucleotide and read the stored data from the target well of the setof target wells that is adjacent to a used well, while leaving thebarrier features deactivated.

Variations on any one or more of the above implementations exist,wherein the method may further comprise, where the target well is notadjacent to any used well of the plurality of wells, providing the readstored data to a requesting device without performing a cross talkquality control check on the read stored data.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein and to achieve thebenefits/advantages as described herein. In particular, all combinationsof claimed subject matter appearing at the end of this disclosure arecontemplated as being part of the inventive subject matter disclosedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

The details of one or more implementations are set forth in theaccompanying drawings and the description below. Other features,aspects, and advantages will become apparent from the description, thedrawings, and the claims, in which:

FIG. 1 depicts a block schematic view of an example of a system that maybe used to conduct biochemical processes;

FIG. 2 depicts a block schematic cross-sectional view of an example of aconsumable cartridge that may be utilized with the system of FIG. 1;

FIG. 3 depicts a perspective view of an example of a flow cell that maybe utilized with the system of FIG. 1;

FIG. 4 depicts an enlarged perspective view of a channel of the flowcell of FIG. 3;

FIG. 5 depicts a block schematic cross-sectional view of an example ofwells that may be incorporated into the channel of FIG. 4;

FIG. 6 depicts a flow chart of an example of a process for readingpolynucleotides;

FIG. 7 depicts a block schematic cross-sectional view of another exampleof wells that may be incorporated into the channel of FIG. 4;

FIG. 8 depicts a flow chart of an example of a process for writingpolynucleotides;

FIG. 9 depicts a top plan view of an example of an electrode assembly;

FIG. 10 depicts a block schematic cross-sectional view of anotherexample of wells that may be incorporated into the channel of FIG. 4;

FIG. 11A depicts a block schematic cross-sectional view of anotherexample of a well that may be incorporated into the channel of FIG. 4,with a valve in a closed state;

FIG. 11B depicts a block schematic cross-sectional view of the well ofFIG. 11A, with the valve in an open state;

FIG. 12 depicts a block schematic cross-sectional view of anotherexample of a well that may be incorporated into the channel of FIG. 4;

FIG. 13 depicts a block schematic cross-sectional view of anotherexample of a well that may be incorporated into the channel of FIG. 4;

FIG. 14 depicts a block schematic cross-sectional view of anotherexample of a well that may be incorporated into the channel of FIG. 4;

FIG. 15 depicts a block schematic cross-sectional view of anotherexample of a well that may be incorporated into the channel of FIG. 4;

FIG. 16A depicts a block schematic cross-sectional view of a flow cellwith a movable barrier positioned between adjacent wells;

FIG. 16B depicts a block schematic cross-sectional view of the flow cellof FIG. 16A with the movable barrier positioned over one of the wells;

FIG. 17 depicts a block schematic cross-sectional view of anotherexample of a flow cell that may be utilized with the system of FIG. 1;

FIG. 18 depicts a block schematic cross-sectional view of an example ofa flow cell that may be utilized with the system of FIG. 1;

FIG. 19 depicts a block schematic cross-sectional view of anotherexample of a flow cell that may be utilized with the system of FIG. 1;

FIG. 20 depicts a block schematic cross-sectional view of anotherexample of a flow cell that may be utilized with the system of FIG. 1;and

FIG. 21 depicts a flow chart showing an example of a method that may beperformed while machine-writing DNA.

It will be recognized that some or all of the figures are schematicrepresentations for purposes of illustration. The figures are providedfor the purpose of illustrating one or more implementations with theexplicit understanding that they will not be used to limit the scope orthe meaning of the claims.

DETAILED DESCRIPTION

In some aspects, methods and systems are disclosed herein forsynthesizing DNA (or other biological material) to store data or otherinformation; and/or reading machine-written DNA (or other biologicalmaterial) to retrieve the machine-written data or other information.Machine-written DNA may provide an alternative to traditional forms ofdata storage (e.g., magnetic storage, optical storage, and solid-statestorage). In some aspects, methods and systems are disclosed herein forpreventing optical cross-talk between adjacent wells of a flow cellwhile synthesizing DNA (or other biological material) to store data orother information; and/or reading machine-written DNA (or otherbiological material) to retrieve the machine-written data or otherinformation. Machine-written DNA may provide faster read-write speeds,longer data retention, reduced power usage, and higher data density.Examples of how digital information may be stored in DNA are disclosedin U.S. Pub. No. 2015/0261664, entitled “High-Capacity of Storage ofDigital Information in DNA,” published Sep. 17, 2015, which isincorporated by reference herein in its entirety. For example, methodsfrom code theory to enhance the recoverability of the encoded messagesfrom the DNA segment, including forbidding DNA homopolymers (i.e. runsof more than one identical base) that are known to be associated withhigher error rates in existing high throughput technologies may be used.Further, an error-detecting component, analogous to a parity-check bit,may be integrated into the indexing information in the code. Morecomplex schemes, including but not limited to error-correcting codesand, indeed, substantially any form of digital data security (e.g.,RAID-based schemes) currently employed in informatics, may beimplemented in future developments of the DNA storage scheme. The DNAencoding of information may be computed using software. The bytescomprising each computer file may be represented by a DNA sequence withno homopolymers by an encoding scheme to produce an encoded file thatreplaces each byte by five or six bases forming the DNA sequence.

The code used in the encoding scheme may be constructed to permit astraightforward encoding that is close to the optimum informationcapacity for a run length-limited channel (e.g., no repeatednucleotides), though other encoding schemes may be used. The resultingin silico DNA sequences may be too long to be readily produced bystandard oligonucleotide synthesis and may be split into overlappingsegments of a length of 100 bases with an overlap of 75 bases. To reducethe risk of systematic synthesis errors introduced to any particular runof bases, alternate ones of the segments may be converted to theirreverse complement, meaning that each base may be “written” four times,twice in each direction. Each segment may then be augmented with anindexing information that permits determination of the computer filefrom which the segment originated and its location within that computerfile, plus simple error-detection information. This indexing informationmay also be encoded in as non-repeating DNA nucleotides and appended tothe information storage bases of the DNA segments. The division of theDNA segments into lengths of 100 bases with an overlap of 75 bases ispurely arbitrary and illustrative, and it is understood that otherlengths and overlaps may be used and is not limiting.

Other encoding schemes for the DNA segments may be used, for example toprovide enhanced error-correcting properties. The amount of indexinginformation may be increased in order to allow more or larger files tobe encoded. One extension to the coding scheme in order to avoidsystematic patterns in the DNA segments may be to add change theinformation. One way may use the “shuffling” of information in the DNAsegments, where the information may be retrieved if one knows thepattern of shuffling. Different patterns of shuffles may be used fordifferent ones of the DNA segments. A further way is to add a degree ofrandomness into the information in each one of the DNA segments. Aseries of random digits may be used for this, using modular addition ofthe series of random digits and the digits comprising the informationencoded in the DNA segments. The information may be retrieved by modularsubtraction during decoding if one knows the series of random digitsused. Different series of random digits may be used for different onesof the DNA segments The data-encoding component of each string maycontain Shannon information at 5.07 bits per DNA base, which is close tothe theoretical optimum of 5.05 bits per DNA base for base-4 channelswith run length limited to one. The indexing implementation may permit314=4782969 unique data locations. Increasing the number of indexingtrits (and therefore bases) used to specify file and intra-file locationby just two, to 16, gives 316=43046721 unique locations, in excess ofthe 16.8M that is the practical maximum for the Nested Primer MolecularMemory (NPMM) scheme.

The DNA segment designs may be synthesized in three distinct runs (withthe DNA segments randomly assigned to runs) to create approx. 1.2×10⁷copies of each DNA segment design. Phosphoramidite chemistry may beused, and inkjet printing and flow cell reactor technologies in anin-situ microarray synthesis platform may be employed. The inkjetprinting within an anhydrous chamber may allow the delivery of verysmall volumes of phosphoramidites to a confined coupling area on a 2Dplanar surface, resulting in the addition of hundreds of thousands ofbases in parallel. Subsequent oxidation and detritylation may be carriedout in a flow cell reactor. Once DNA synthesis is completed, theoligonucleotides may then be cleaved from the surface and deprotected.

Adapters may then be added to the DNA segments to enable a plurality ofcopies of the DNA segments to be made. A DNA segment with no adapter mayrequire additional chemical processes to “kick start” the chemistry forthe synthesis of the multiple copies by adding additional groups ontothe ends of the DNA segments. Oligonucleotides may be amplified usingpolymerase chain reaction (PCR) methods and paired-end PCR primers,followed by bead purification and quantification. Oligonucleotides maythen be sequenced to produce reads of 104 bases. The digital informationdecoding may then be carried out via sequencing of the central bases ofeach oligo from both ends and rapid computation of full-length oligosand removal of sequence reads inconsistent with the designs. Sequencereads may be decoded using computer software that exactly reverses theencoding process. Sequence reads for which the parity-check tritindicates an error or that may be unambiguously decoded or assigned to areconstructed computer file may be discarded. Locations within everydecoded file may be detected in multiple different sequenced DNA oligos,and simple majority voting may be used to resolve any discrepanciescaused by the DNA synthesis or the sequencing errors.

While several examples herein are provided in the context ofmachine-written DNA, it is contemplated that the principles describedherein may be applied to other kinds of machine-written biologicalmaterial.

As used herein, the term “machine-written DNA” shall be read to includeone or more strands of polynucleotides that are generated by a machine,or otherwise modified by a machine, to store data or other information.One example of the polynucleotide herein is a DNA. It is noted thatwhile the term “DNA” in the context of DNA being read or written is usedthroughout this disclosure, the term is used only as a representativeexample of a polynucleotide and may encompass the concept of apolynucleotide. “Machine,” as used herein in reference to“machine-written,” may include an instrument or system speciallydesigned for writing DNA as described in greater detail herein. Thesystem may be non-biological or biological. In one example, thebiological system may comprise, or is, a polymerase. For example, thepolymerase may be terminal deoxynucleotidyl transferase (TdT). In abiological system, the process may be additionally controlled by amachine hardware (e.g., processor) or an algorithm. “Machine-writtenDNA” may include any polynucleotide having one or more base sequenceswritten by a machine. While machine-written DNA is used herein as anexample, other polynucleotide strands may be substituted formachine-written DNA described herein. “Machine-written DNA” may includenatural bases and modifications of natural bases, including but notlimited to bases modified with methylation or other chemical tags; anartificially synthesized polymer that is similar to DNA, such as peptidenucleic acid (PNA); or Morpholino DNA. “Machine-written DNA” may alsoinclude DNA strands or other polynucleotides that are formed by at leastone strand of bases originating from nature (e.g., extracted from anaturally occurring organism), with a machine-written strand of basessecured thereto either in a parallel fashion or in an end-to-endfashion. In other implementations, “machine-written DNA” may be writtenby a biological system (e.g., enzyme) in lieu of, or in addition to, anon-biological system (e.g., the electrode machine) writing of DNAdescribed herein. In other words, “machine-written DNA” may be writtendirectly by a machine; or by an enzyme (e.g., polymerase) that iscontrolled by an algorithm and/or machine.

“Machine-written DNA” may include data that have been converted from araw form (e.g., a photograph, a text document, etc.) into a binary codesequence using known techniques, with that binary code sequence thenbeing converted to a DNA base sequence using known techniques, and withthat DNA base sequence then being generated by a machine in the form ofone or more DNA strands or other polynucleotides. Alternatively,“machine-written DNA” may be generated to index or otherwise trackpre-existing DNA, to store data or information from any other source andfor any suitable purpose, without necessarily requiring an intermediatestep of converting raw data to a binary code.

As described in greater detail below, machine-written DNA may be writtento and/or read from a reaction site. As used herein, the term “reactionsite” is a localized region where at least one designated reaction mayoccur. A reaction site may include support surfaces of a reactionstructure or substrate where a substance may be immobilized thereon. Forinstance, the reaction site may be a discrete region of space where adiscrete group of DNA strands or other polynucleotides are written. Thereaction site may permit chemical reactions that are isolated fromreactions that are in adjacent reaction sites. Devices that providemachine-writing of DNA may include flow cells with wells having writingfeatures (e.g., electrodes) and/or reading features. In some instances,the reaction site may include a surface of a reaction structure (whichmay be positioned in a channel of a flow cell) that already has areaction component thereon, such as a colony of polynucleotides thereon.In some flow cells, the polynucleotides in the colony have the samesequence, being for example, clonal copies of a single stranded ordouble stranded template. However, in some flow cells a reaction sitemay contain only a single polynucleotide molecule, for example, in asingle stranded or double stranded form.

A plurality of reaction sites may be randomly distributed along thereaction structure of the flow cells or may be arranged in apredetermined manner (e.g., side-by-side in a matrix, such as inmicroarrays). A reaction site may also include a reaction chamber,recess, or well that at least partially defines a spatial region orvolume configured to compartmentalize the designated reaction. As usedherein, the term “reaction chamber” or “reaction recess” includes adefined spatial region of the support structure (which is oftenfluidically coupled with a flow channel). A reaction recess may be atleast partially separated from the surrounding environment or otherspatial regions. For example, a plurality of reaction recesses may beseparated from each other by shared walls. As a more specific example,the reaction recesses may be nanowells comprising an indent, pit, well,groove, cavity or depression defined by interior surfaces of a detectionsurface and have an opening or aperture (i.e., be open-sided) so thatthe nanowells may be fluidically coupled with a flow channel.

A plurality of reaction sites may be randomly distributed along thereaction structure of the flow cells or may be arranged in apredetermined manner (e.g., side-by-side in a matrix, such as inmicroarrays). A reaction site may also include a reaction chamber,recess, or well that at least partially defines a spatial region orvolume configured to compartmentalize the designated reaction. As usedherein, the term “reaction chamber” or “reaction recess” includes adefined spatial region of the support structure (which is oftenfluidically coupled with a flow channel). A reaction recess may be atleast partially separated from the surrounding environment or otherspatial regions. For example, a plurality of reaction recesses may beseparated from each other by shared walls. As a more specific example,the reaction recesses may be nanowells comprising an indent, pit, well,groove, cavity or depression defined by interior surfaces of a detectionsurface and have an opening or aperture (i.e., be open-sided) so thatthe nanowells may be fluidically coupled with a flow channel.

To read the machine-written DNA, one or more discrete detectable regionsof reaction sites may be defined. Such detectable regions may beimageable regions, electrical detection regions, or other types ofregions that may have a measurable change in a property (or absence ofchange in the property) based on the type of nucleotide present duringthe reading process.

As used herein, the term “pixel” refers to a discrete imageable region.Each imageable region may include a compartment or discrete region ofspace where a polynucleotide is present. In some instances, a pixel mayinclude two or more reaction sites (e.g., two or more reaction chambers,two or more reaction recesses, two or more wells, etc.). In some otherinstances, a pixel may include just one reaction site. Each pixel isdetected using a corresponding detection device, such as an image sensoror other light detection device. The light detection device may bemanufactured using integrated circuit manufacturing processes, such asprocesses used to manufacture charged-coupled devices circuits (CCD) orcomplementary-metal-oxide semiconductor (CMOS) devices or circuits. Thelight detection device may thereby include, for example, one or moresemiconductor materials, and may take the form of, for example, a CMOSlight detection device (e.g., a CMOS image sensor) or a CCD imagesensor, another type of image sensor. A CMOS image sensor may include anarray of light sensors (e.g. photodiodes). In one implementation, asingle image sensor may be used with an objective lens to captureseveral “pixels,” during an imaging event. In some otherimplementations, each discrete photodiode or light sensor may capture acorresponding pixel. In some implementations, light sensors (e.g.,photodiodes) of one or more detection devices may be associated withcorresponding reaction sites. A light sensor that is associated with areaction site may detect light emissions from the associated reactionsite. In some implementations, the detection of light emissions may bedone via at least one light guide when a designated reaction hasoccurred at the associated reaction site. In some implementations, aplurality of light sensors (e.g., several pixels of a light detection orcamera device) may be associated with a single reaction site. In someimplementations, a single light sensor (e.g. a single pixel) may beassociated with a single reaction site or with a group of reactionsites.

As used herein, the term “synthesis” shall be read to include processeswhere DNA is generated by a machine to store data or other information.Thus, machine-written DNA may constitute synthesized DNA. As usedherein, the terms “consumable cartridge,” “reagent cartridge,”“removeable cartridge,” and/or “cartridge” refer to the same cartridgeand/or a combination of components making an assembly for a cartridge orcartridge system. The cartridges described herein may be independent ofthe element with the reaction sites, such as a flow cell having aplurality of wells. In some instances, a flow cell may be removablyinserted into a cartridge, which is then inserted into an instrument. Insome other implementations, the flow cell may be removably inserted intothe instrument without a cartridge. As used herein, the term“biochemical analysis” may include at least one of biological analysisor chemical analysis.

The term “based on” should be understood to mean that something isdetermined at least in part by the thing it is indicated as being “basedon.” To indicate that something must necessarily be completelydetermined by something else, it is described as being based exclusivelyon whatever it is completely determined by.

The term “non-nucleotide memory” should be understood to refer to anobject, device or combination of devices capable of storing data orinstructions in a form other than nucleotides that may be retrievedand/or processed by a device. Examples of “non-nucleotide memory”include solid state memory, magnetic memory, hard drives, optical drivesand combinations of the foregoing (e.g., magneto-optical storageelements).

The term “DNA storage device” should be understood to refer to anobject, device, or combination of devices configured to store data orinstructions in the form of sequences of polynucleotides such asmachine-written DNA. Examples of “DNA storage devices” include flowcells having addressable wells as described herein, systems comprisingmultiple such flow cells, and tubes or other containers storingnucleotide sequences that have been cleaved from the surface on whichthey were synthesized. As used herein, the term “nucleotide sequence” or“polynucleotide sequence” should be read to include a polynucleotidemolecule, as well as the underlying sequence of the molecule, dependingon context. A sequence of a polynucleotide may contain (or encode)information indicative of certain physical characteristics.

Implementations set forth herein may be used to perform designatedreactions for consumable cartridge preparation and/or biochemicalanalysis and/or synthesis of machine-written DNA.

I. System Overview

FIG. 1 is a schematic diagram of a system 100 that is configured toconduct biochemical analysis and/or synthesis. The system 100 mayinclude a base instrument 102 that is configured to receive andseparably engage a removable cartridge 200 and/or a component with oneor more reaction sites. The base instrument 102 and the removablecartridge 200 may be configured to interact with each other to transporta biological material to different locations within the system 100and/or to conduct designated reactions that include the biologicalmaterial in order to prepare the biological material for subsequentanalysis (e.g., by synthesizing the biological material), and,optionally, to detect one or more events with the biological material.In some implementations, the base instrument 102 may be configured todetect one or more events with the biological material directly on theremovable cartridge 200. The events may be indicative of a designatedreaction with the biological material. The removable cartridge 200 maybe constructed according to any of the cartridges described herein.

Although the following is with reference to the base instrument 102 andthe removable cartridge 200 as shown in FIG. 1, it is understood thatthe base instrument 102 and the removable cartridge 200 illustrate onlyone implementation of the system 100 and that other implementationsexist. For example, the base instrument 102 and the removable cartridge200 include various components and features that, collectively, executeseveral operations for preparing the biological material and/oranalyzing the biological material. Moreover, although the removablecartridge 200 described herein includes an element with the reactionsites, such as a flow cell having a plurality of wells, other cartridgesmay be independent of the element with the reaction sites and theelement with the reaction sites may be separately insertable into thebase instrument 102. That is, in some instances a flow cell may beremovably inserted into the removable cartridge 200, which is theninserted into the base instrument 102. In some other implementations,the flow cell may be removably inserted directly into the baseinstrument 102 without the removable cartridge 200. In still furtherimplementations, the flow cell may be integrated into the removablecartridge 200 that is inserted into the base instrument 102.

In the illustrated implementation, each of the base instrument 102 andthe removable cartridge 200 are capable of performing certain functions.It is understood, however, that the base instrument 102 and theremovable cartridge 200 may perform different functions and/or may sharesuch functions. For example, the base instrument 102 is shown to includea detection assembly 110 (e.g., an imaging device) that is configured todetect the designated reactions at the removable cartridge 200. Inalternative implementations, the removable cartridge 200 may include thedetection assembly and may be communicatively coupled to one or morecomponents of the base instrument 102. As another example, the baseinstrument 102 is a “dry” instrument that does not provide, receive, orexchange liquids with the removable cartridge 200. That is, as shown,the removable cartridge 200 includes a consumable reagent portion 210and a flow cell receiving portion 220. The consumable reagent portion210 may contain reagents used during biochemical analysis and/orsynthesis. The flow cell receiving portion 220 may include an opticallytransparent region or other detectible region for the detection assembly110 to perform detection of one or more events occurring within the flowcell receiving portion 220. In alternative implementations, the baseinstrument 102 may provide, for example, reagents or other liquids tothe removable cartridge 200 that are subsequently consumed (e.g., usedin designated reactions or synthesis procedures) by the removablecartridge 200.

As used herein, the biological material may include one or morebiological or chemical substances, such as nucleosides, nucleotides,nucleic acids, polynucleotides, oligonucleotides, proteins, enzymes,peptides, oligopeptides, polypeptides, antibodies, antigens, ligands,receptors, polysaccharides, carbohydrates, polyphosphates, nanopores,organelles, lipid layers, cells, tissues, organisms, and/or biologicallyactive chemical compound(s), such as analogs or mimetics of theaforementioned species. In some instances, the biological material mayinclude whole blood, lymphatic fluid, serum, plasma, sweat, tear,saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid,vaginal excretion, serous fluid, synovial fluid, pericardial fluid,peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid,bile, urine, gastric fluid, intestinal fluid, fecal samples, liquidscontaining single or multiple cells, liquids containing organelles,fluidized tissues, fluidized organisms, viruses including viralpathogens, liquids containing multi-celled organisms, biological swabsand biological washes. In some instances, the biological material mayinclude a set of synthetic sequences, including but not limited tomachine-written DNA, which may be fixed (e.g., attached in specificwells in a cartridge) or unfixed (e.g., stored in a tube).

In some implementations, the biological material may include an addedmaterial, such as water, deionized water, saline solutions, acidicsolutions, basic solutions, detergent solutions and/or pH buffers. Theadded material may also include reagents that will be used during thedesignated assay protocol to conduct the biochemical reactions. Forexample, added liquids may include material to conduct multiplepolymerase-chain-reaction (PCR) cycles with the biological material. Inother aspects, the added material may be a carrier for the biologicalmaterial such as cell culture media or other buffered and/or pH adjustedand/or isotonic carrier that may allow for or preserve the biologicalfunction of the biological material.

It should be understood, however, that the biological material that isanalyzed may be in a different form or state than the biologicalmaterial loaded into or created by the system 100. For example, abiological material loaded into the system 100 may include whole bloodor saliva or cell population that is subsequently treated (e.g., viaseparation or amplification procedures) to provide prepared nucleicacids. The prepared nucleic acids may then be analyzed (e.g., quantifiedby PCR or sequenced by SBS) by the system 100. Accordingly, when theterm “biological material” is used while describing a first operation,such as PCR, and used again while describing a subsequent secondoperation, such as sequencing, it is understood that the biologicalmaterial in the second operation may be modified with respect to thebiological material prior to or during the first operation. For example,sequencing (e.g. SBS) may be carried out on amplicon nucleic acids thatare produced from template nucleic acids that are amplified in a prioramplification (e.g. PCR). In this case the amplicons are copies of thetemplates and the amplicons are present in higher quantity compared tothe quantity of the templates.

In some implementations, the system 100 may automatically prepare asample for biochemical analysis based on a substance provided by theuser (e.g., whole blood or saliva or a population of cells). However, inother implementations, the system 100 may analyze biological materialsthat are partially or preliminarily prepared for analysis by the user.For example, the user may provide a solution including nucleic acidsthat were already isolated and/or amplified from whole blood; or mayprovide a virus sample in which the RNA or DNA sequence is partially orwholly exposed for processing.

As used herein, a “designated reaction” includes a change in at leastone of a chemical, electrical, physical, or optical property (orquality) of an analyte-of-interest. In particular implementations, thedesignated reaction is an associative binding event (e.g., incorporationof a fluorescently labeled biomolecule with the analyte-of-interest).The designated reaction may be a dissociative binding event (e.g.,release of a fluorescently labeled biomolecule from ananalyte-of-interest). The designated reaction may be a chemicaltransformation, chemical change, or chemical interaction. The designatedreaction may also be a change in electrical properties. For example, thedesignated reaction may be a change in ion concentration within asolution. Some reactions include, but are not limited to, chemicalreactions such as reduction, oxidation, addition, elimination,rearrangement, esterification, amidation, etherification, cyclization,or substitution; binding interactions in which a first chemical binds toa second chemical; dissociation reactions in which two or more chemicalsdetach from each other; fluorescence; luminescence; bioluminescence;chemiluminescence; and biological reactions, such as nucleic acidreplication, nucleic acid amplification, nucleic acid hybridization,nucleic acid ligation, phosphorylation, enzymatic catalysis, receptorbinding, or ligand binding. The designated reaction may also be additionor removal of a proton, for example, detectable as a change in pH of asurrounding solution or environment. An additional designated reactionmay be detecting the flow of ions across a membrane (e.g., natural orsynthetic bilayer membrane). For example, as ions flow through amembrane, the current is disrupted, and the disruption may be detected.Field sensing of charged tags may also be used; as may thermal sensingand other suitable analytical sensing techniques.

In particular implementations, the designated reaction includes theincorporation of a fluorescently labeled molecule to an analyte. Theanalyte may be an oligonucleotide and the fluorescently labeled moleculemay be a nucleotide. The designated reaction may be detected when anexcitation light is directed toward the oligonucleotide having thelabeled nucleotide, and the fluorophore emits a detectable fluorescentsignal. In alternative implementations, the detected fluorescence is aresult of chemiluminescence and/or bioluminescence. A designatedreaction may also increase fluorescence (or Forster) resonance energytransfer (FRET), for example, by bringing a donor fluorophore inproximity to an acceptor fluorophore, decrease FRET by separating donorand acceptor fluorophores, increase fluorescence by separating aquencher from a fluorophore or decrease fluorescence by co-locating aquencher and fluorophore.

As used herein, a “reaction component” includes any substance that maybe used to obtain a designated reaction. For example, reactioncomponents include reagents, catalysts such as enzymes, reactants forthe reaction, samples, products of the reaction, other biomolecules,salts, metal cofactors, chelating agents, and buffer solutions (e.g.,hydrogenation buffer). The reaction components may be delivered,individually in solutions or combined in one or more mixture, to variouslocations in a fluidic network. For instance, a reaction component maybe delivered to a reaction chamber where the biological material isimmobilized. The reaction components may interact directly or indirectlywith the biological material. In some implementations, the removablecartridge 200 is preloaded with one or more of the reaction componentsinvolved in carrying out a designated assay protocol. Preloading mayoccur at one location (e.g. a manufacturing facility) prior to receiptof the cartridge 200 by a user (e.g. at a customer's facility). Forexample, the one or more reaction components or reagents may bepreloaded into the consumable reagent portion 210. In someimplementations, the removable cartridge 200 may also be preloaded witha flow cell in the flow cell receiving portion 220.

In some implementations, the base instrument 102 may be configured tointeract with one removable cartridge 200 per session. After thesession, the removable cartridge 200 may be replaced with anotherremovable cartridge 200. In other implementations, the base instrument102 may be configured to interact with more than one removable cartridge200 per session. As used herein, the term “session” includes performingat least one of sample preparation and/or biochemical analysis protocol.Sample preparation may include synthesizing the biological material;and/or separating, isolating, modifying, and/or amplifying one or morecomponents of the biological material so that the prepared biologicalmaterial is suitable for analysis. In some implementations, a sessionmay include continuous activity in which a number of controlledreactions are conducted until (a) a designated number of reactions havebeen conducted, (b) a designated number of events have been detected,(c) a designated period of system time has elapsed, (d) signal-to-noisehas dropped to a designated threshold; (e) a target component has beenidentified; (f) system failure or malfunction has been detected; and/or(g) one or more of the resources for conducting the reactions hasdepleted. Alternatively, a session may include pausing system activityfor a period of time (e.g., minutes, hours, days, weeks) and latercompleting the session until at least one of (a)-(g) occurs.

An assay protocol may include a sequence of operations for conductingthe designated reactions, detecting the designated reactions, and/oranalyzing the designated reactions. Collectively, the removablecartridge 200 and the base instrument 102 may include the components forexecuting the different operations. The operations of an assay protocolmay include fluidic operations, thermal-control operations, detectionoperations, and/or mechanical operations.

A fluidic operation includes controlling the flow of fluid (e.g., liquidor gas) through the system 100, which may be actuated by the baseinstrument 102 and/or by the removable cartridge 200. In one example,the fluid is in liquid form. For example, a fluidic operation mayinclude controlling a pump to induce flow of the biological material ora reaction component into a reaction chamber.

A thermal-control operation may include controlling a temperature of adesignated portion of the system 100, such as one or more portions ofthe removable cartridge 200. By way of example, a thermal-controloperation may include raising or lowering a temperature of a polymerasechain reaction (PCR) zone where a liquid that includes the biologicalmaterial is stored.

A detection operation may include controlling activation of a detectoror monitoring activity of the detector to detect predeterminedproperties, qualities, or characteristics of the biological material. Asone example, the detection operation may include capturing images of adesignated area that includes the biological material to detectfluorescent emissions from the designated area. The detection operationmay include controlling a light source to illuminate the biologicalmaterial or controlling a detector to observe the biological material.

A mechanical operation may include controlling a movement or position ofa designated component. For example, a mechanical operation may includecontrolling a motor to move a valve-control component in the baseinstrument 102 that operably engages a movable valve in the removablecartridge 200. In some cases, a combination of different operations mayoccur concurrently. For example, the detector may capture images of thereaction chamber as the pump controls the flow of fluid through thereaction chamber. In some cases, different operations directed towarddifferent biological materials may occur concurrently. For instance, afirst biological material may be undergoing amplification (e.g., PCR)while a second biological material may be undergoing detection.

Similar or identical fluidic elements (e.g., channels, ports,reservoirs, etc.) may be labeled differently to more readily distinguishthe fluidic elements. For example, ports may be referred to as reservoirports, supply ports, network ports, feed port, etc. It is understoodthat two or more fluidic elements that are labeled differently (e.g.,reservoir channel, sample channel, flow channel, bridge channel) do notrequire that the fluidic elements be structurally different. Moreover,the claims may be amended to add such labels to more readily distinguishsuch fluidic elements in the claims.

A “liquid,” as used herein, is a substance that is relativelyincompressible and has a capacity to flow and to conform to a shape of acontainer or a channel that holds the substance. A liquid may beaqueous-based and include polar molecules exhibiting surface tensionthat holds the liquid together. A liquid may also include non-polarmolecules, such as in an oil-based or non-aqueous substance. It isunderstood that references to a liquid in the present application mayinclude a liquid comprising the combination of two or more liquids. Forexample, separate reagent solutions may be later combined to conductdesignated reactions.

One or more implementations may include retaining the biologicalmaterial (e.g., template nucleic acid) at a designated location wherethe biological material is analyzed. As used herein, the term“retained,” when used with respect to a biological material, includesattaching the biological material to a surface or confining thebiological material within a designated space. As used herein, the term“immobilized,” when used with respect to a biological material, includesattaching the biological material to a surface in or on a solid support.Immobilization may include attaching the biological material at amolecular level to the surface. For example, a biological material maybe immobilized to a surface of a substrate using adsorption techniquesincluding non-covalent interactions (e.g., electrostatic forces, van derWaals, and dehydration of hydrophobic interfaces) and covalent bindingtechniques where functional groups or linkers facilitate attaching thebiological material to the surface. Immobilizing a biological materialto a surface of a substrate may be based upon the properties of thesurface of the substrate, the liquid medium carrying the biologicalmaterial, and the properties of the biological material itself. In somecases, a substrate surface may be functionalized (e.g., chemically orphysically modified) to facilitate immobilizing the biological materialto the substrate surface. The substrate surface may be first modified tohave functional groups bound to the surface. The functional groups maythen bind to the biological material to immobilize the biologicalmaterial thereon. In some cases, a biological material may beimmobilized to a surface via a gel.

In some implementations, nucleic acids may be immobilized to a surfaceand amplified using bridge amplification. Another useful method foramplifying nucleic acids on a surface is rolling circle amplification(RCA), for example, using methods set forth in further detail below. Insome implementations, the nucleic acids may be attached to a surface andamplified using one or more primer pairs. For example, one of theprimers may be in solution and the other primer may be immobilized onthe surface (e.g., 5′-attached). By way of example, a nucleic acidmolecule may hybridize to one of the primers on the surface followed byextension of the immobilized primer to produce a first copy of thenucleic acid. The primer in solution then hybridizes to the first copyof the nucleic acid which may be extended using the first copy of thenucleic acid as a template. Optionally, after the first copy of thenucleic acid is produced, the original nucleic acid molecule mayhybridize to a second immobilized primer on the surface and may beextended at the same time or after the primer in solution is extended.In any implementation, repeated rounds of extension (e.g.,amplification) using the immobilized primer and primer in solution maybe used to provide multiple copies of the nucleic acid. In someimplementations, the biological material may be confined within apredetermined space with reaction components that are configured to beused during amplification of the biological material (e.g., PCR).

One or more implementations set forth herein may be configured toexecute an assay protocol that is or includes an amplification (e.g.,PCR) protocol. During the amplification protocol, a temperature of thebiological material within a reservoir or channel may be changed inorder to amplify a target sequence or the biological material (e.g., DNAof the biological material). By way of example, the biological materialmay experience (1) a pre-heating stage of about 95° C. for about 75seconds; (2) a denaturing stage of about 95° C. for about 15 seconds;(3) an annealing-extension stage of about of about 59° C. for about 45seconds; and (4) a temperature holding stage of about 72° C. for about60 seconds. Implementations may execute multiple amplification cycles.It is noted that the above cycle describes only one particularimplementation and that alternative implementations may includemodifications to the amplification protocol.

The methods and systems set forth herein may use arrays having featuresat any of a variety of densities including, for example, at least about10 features/cm², about 100 features/cm², about 500 features/cm², about1,000 features/cm², about 5,000 features/cm², about 10,000 features/cm²,about 50,000 features/cm², about 100,000 features/cm², about 1,000,000features/cm², about 5,000,000 features/cm², or higher. The methods andapparatus set forth herein may include detection components or deviceshaving a resolution that is at least sufficient to resolve individualfeatures at one or more of these densities.

The base instrument 102 may include a user interface 130 that isconfigured to receive user inputs for conducting a designated assayprotocol and/or configured to communicate information to the userregarding the assay. The user interface 130 may be incorporated with thebase instrument 102. For example, the user interface 130 may include atouchscreen that is attached to a housing of the base instrument 102 andconfigured to identify a touch from the user and a location of the touchrelative to information displayed on the touchscreen. Alternatively, theuser interface 130 may be located remotely with respect to the baseinstrument 102.

II. Cartridge

The removable cartridge 200 is configured to separably engage orremovably couple to the base instrument 102 at a cartridge chamber 140.As used herein, when the terms “separably engaged” or “removablycoupled” (or the like) are used to describe a relationship between aremovable cartridge 200 and a base instrument 102. The term is intendedto mean that a connection between the removable cartridge 200 and thebase instrument 102 are separable without destroying the base instrument102. Accordingly, the removable cartridge 200 may be separably engagedto the base instrument 102 in an electrical manner such that theelectrical contacts of the base instrument 102 are not destroyed. Theremovable cartridge 200 may be separably engaged to the base instrument102 in a mechanical manner such that features of the base instrument 102that hold the removable cartridge 200, such as the cartridge chamber140, are not destroyed. The removable cartridge 200 may be separablyengaged to the base instrument 102 in a fluidic manner such that theports of the base instrument 102 are not destroyed. The base instrument102 is not considered to be “destroyed,” for example, if only a simpleadjustment to the component (e.g., realigning) or a simple replacement(e.g., replacing a nozzle) is required. Components (e.g., the removablecartridge 200 and the base instrument 102) may be readily separable whenthe components may be separated from each other without undue effort ora significant amount of time spent in separating the components. In someimplementations, the removable cartridge 200 and the base instrument 102may be readily separable without destroying either the removablecartridge 200 or the base instrument 102.

In some implementations, the removable cartridge 200 may be permanentlymodified or partially damaged during a session with the base instrument102. For instance, containers holding liquids may include foil coversthat are pierced to permit the liquid to flow through the system 100. Insuch implementations, the foil covers may be damaged such that thedamaged container is to be replaced with another container. Inparticular implementations, the removable cartridge 200 is a disposablecartridge such that the removable cartridge 200 may be replaced andoptionally disposed after a single use. Similarly, a flow cell of theremovable cartridge 200 may be separately disposable such that the flowcell may be replaced and optionally disposed after a single use.

In other implementations, the removable cartridge 200 may be used formore than one session while engaged with the base instrument 102 and/ormay be removed from the base instrument 102, reloaded with reagents, andre-engaged to the base instrument 102 to conduct additional designatedreactions. Accordingly, the removable cartridge 200 may be refurbishedin some cases such that the same removable cartridge 200 may be usedwith different consumables (e.g., reaction components and biologicalmaterials). Refurbishing may be carried out at a manufacturing facilityafter the cartridge 200 has been removed from a base instrument 102located at a customer's facility.

The cartridge chamber 140 may include a slot, mount, connectorinterface, and/or any other feature to receive the removable cartridge200 or a portion thereof to interact with the base instrument 102.

The removable cartridge 200 may include a fluidic network that may holdand direct fluids (e.g., liquids or gases) therethrough. The fluidicnetwork may include a plurality of interconnected fluidic elements thatare capable of storing a fluid and/or permitting a fluid to flowtherethrough. Non-limiting examples of fluidic elements includechannels, ports of the channels, cavities, storage devices, reservoirsof the storage devices, reaction chambers, waste reservoirs, detectionchambers, multipurpose chambers for reaction and detection, and thelike. For example, the consumable reagent portion 210 may include one ormore reagent wells or chambers storing reagents and may be part of orcoupled to the fluidic network. The fluidic elements may be fluidicallycoupled to one another in a designated manner so that the system 100 iscapable of performing sample preparation and/or analysis.

As used herein, the term “fluidically coupled” (or like term) refers totwo spatial regions being connected together such that a liquid or gasmay be directed between the two spatial regions. In some cases, thefluidic coupling permits a fluid to be directed back and forth betweenthe two spatial regions. In other cases, the fluidic coupling isuni-directional such that there is only one direction of flow betweenthe two spatial regions. For example, an assay reservoir may befluidically coupled with a channel such that a liquid may be transportedinto the channel from the assay reservoir. However, in someimplementations, it may not be possible to direct the fluid in thechannel back to the assay reservoir. In particular implementations, thefluidic network may be configured to receive a biological material anddirect the biological material through sample preparation and/or sampleanalysis. The fluidic network may direct the biological material andother reaction components to a waste reservoir.

FIG. 2 depicts an implementation of a consumable cartridge 300. Theconsumable cartridge may be part of a combined removable cartridge, suchas consumable reagent portion 210 of removable cartridge 200 of FIG. 1;or may be a separate reagent cartridge. The consumable cartridge 300 mayinclude a housing 302 and a top 304. The housing 302 may comprise anon-conductive polymer or other material and be formed to make one ormore reagent chambers 310, 320, 330. The reagent chambers 310, 320, 330may be varying in size to accommodate varying volumes of reagents to bestored therein. For instance, a first chamber 310 may be larger than asecond chamber 320, and the second chamber 320 may be larger than athird chamber 330. The first chamber 310 is sized to accommodate alarger volume of a particular reagent, such as a buffer reagent. Thesecond chamber 320 may be sized to accommodate a smaller volume ofreagent than the first chamber 310, such as a reagent chamber holding acleaving reagent. The third chamber 330 may be sized to accommodate aneven smaller volume of reagent than the first chamber 310 and the secondchamber 320, such as a reagent chamber holding a fully functionalnucleotide containing reagent.

In the illustrated implementation, the housing 302 has a plurality ofhousing walls or sides 350 forming the chambers 310, 320, 330 therein.In the illustrated implementation, the housing 302 forms a structurethat is at least substantially unitary or monolithic. In alternativeimplementations, the housing 302 may be constructed by one or moresub-components that are combined to form the housing 302, such asindependently formed compartments for chambers 310, 320, and 330.

The housing 302 may be sealed by the top 304 once reagents are providedinto the respective chambers 310, 320, 330. The top 304 may comprise aconductive or non-conductive material. For instance, the top 304 may bean aluminum foil seal that is adhesively coupled to top surfaces of thehousing 302 to seal the reagents within their respective chambers 310,320, 330. In other implementations, the top 304 may be a plastic sealthat is adhesively coupled to top surfaces of the housing 302 to sealthe reagents within their respective chambers 310, 320, 330.

In some implementations, the housing 302 may also include an identifier390. The identifier 390 may be a radio-frequency identification (RFID)transponder, a barcode, an identification chip, and/or other identifier.In some implementations, the identifier 390 may be embedded in thehousing 302 or attached to an exterior surface. The identifier 390 mayinclude data for a unique identifier for the consumable cartridge 300and/or data for a type of the consumable cartridge 300. The data of theidentifier 390 may be read by the base instrument 102 or a separatedevice configured for warming the consumable cartridge 300, as describedherein.

In some implementations, the consumable cartridge 300 may include othercomponents, such as valves, pumps, fluidic lines, ports, etc. In someimplementations, the consumable cartridge 300 may be contained within afurther exterior housing.

III. System Controller

The base instrument 102 may also include a system controller 120 that isconfigured to control operation of at least one of the removablecartridge 200 and/or the detection assembly 110. The system controller120 may be implemented utilizing any combination of dedicated hardwarecircuitry, boards, DSPs, processors, etc. Alternatively, the systemcontroller 120 may be implemented utilizing an off-the-shelf PC with asingle processor or multiple processors, with the functional operationsdistributed between the processors. As a further option, the systemcontroller 120 may be implemented utilizing a hybrid configuration inwhich certain modular functions are performed utilizing dedicatedhardware, while the remaining modular functions are performed utilizingan off-the-shelf PC and the like.

The system controller 120 may include a plurality of circuitry modulesthat are configured to control operation of certain components of thebase instrument 102 and/or the removable cartridge 200. The term“module” herein may refer to a hardware device configured to performspecific task(s). For instance, the circuitry modules may include aflow-control module that is configured to control flow of fluids throughthe fluidic network of the removable cartridge 200. The flow-controlmodule may be operably coupled to valve actuators and/or s system pump.The flow-control module may selectively activate the valve actuatorsand/or the system pump to induce flow of fluid through one or more pathsand/or to block flow of fluid through one or more paths.

The system controller 120 may also include a thermal-control module. Thethermal-control module may control a thermocycler or other thermalcomponent to provide and/or remove thermal energy from asample-preparation region of the removable cartridge 200 and/or anyother region of the removeable cartridge 200. In one particular example,a thermocycler may increase and/or decrease a temperature that isexperienced by the biological material in accordance with a PCRprotocol.

The system controller 120 may also include a detection module that isconfigured to control the detection assembly 110 to obtain dataregarding the biological material. The detection module may controloperation of the detection assembly 110 either through a direct wiredconnection or through the contact array if the detection assembly 110 ispart of the removable cartridge 200. The detection module may controlthe detection assembly 110 to obtain data at predetermined times or forpredetermined time periods. By way of example, the detection module maycontrol the detection assembly 110 to capture an image of a reactionchamber of the flow cell receiving portion 220 of the removablecartridge when the biological material has a fluorophore attachedthereto. In some implementations, a plurality of images may be obtained.

Optionally, the system controller 120 may include an analysis modulethat is configured to analyze the data to provide at least partialresults to a user of the system 100. For example, the analysis modulemay analyze the imaging data provided by the detection assembly 110. Theanalysis may include identifying a sequence of nucleic acids of thebiological material.

The system controller 120 and/or the circuitry modules described abovemay include one or more logic-based devices, including one or moremicrocontrollers, processors, reduced instruction set computers (RISC),application specific integrated circuits (ASICs), field programmablegate array (FPGAs), logic circuits, and any other circuitry capable ofexecuting functions described herein. In an implementation, the systemcontroller 120 and/or the circuitry modules execute a set ofinstructions that are stored in a computer- or machine-readable mediumtherein in order to perform one or more assay protocols and/or otheroperations. The set of instructions may be stored in the form ofinformation sources or physical memory elements within the baseinstrument 102 and/or the removable cartridge 200. The protocolsperformed by the system 100 may be used to carry out, for example,machine-writing DNA or otherwise synthesizing DNA (e.g., convertingbinary data into a DNA sequence and then synthesizing DNA strands orother polynucleotides representing the binary data), quantitativeanalysis of DNA or RNA, protein analysis, DNA sequencing (e.g.,sequencing-by-synthesis (SBS)), sample preparation, and/or preparationof fragment libraries for sequencing.

The set of instructions may include various commands that instruct thesystem 100 to perform specific operations such as the methods andprocesses of the various implementations described herein. The set ofinstructions may be in the form of a software program. As used herein,the terms “software” and “firmware” are interchangeable and include anycomputer program stored in memory for execution by a computer, includingRAM memory, ROM memory, EPROM memory, EEPROM memory, and non-volatileRAM (NVRAM) memory. The above memory types are only examples and arethus not limiting as to the types of memory usable for storage of acomputer program.

The software may be in various forms such as system software orapplication software. Further, the software may be in the form of acollection of separate programs, or a program module within a largerprogram or a portion of a program module. The software also may includemodular programming in the form of object-oriented programming. Afterobtaining the detection data, the detection data may be automaticallyprocessed by the system 100, processed in response to user inputs, orprocessed in response to a request made by another processing machine(e.g., a remote request through a communication link).

The system controller 120 may be connected to the other components orsub-systems of the system 100 via communication links, which may behardwired or wireless. The system controller 120 may also becommunicatively connected to off-site systems or servers. The systemcontroller 120 may receive user inputs or commands, from a userinterface 130. The user interface 130 may include a keyboard, mouse, atouch-screen panel, and/or a voice recognition system, and the like.

The system controller 120 may serve to provide processing capabilities,such as storing, interpreting, and/or executing software instructions,as well as controlling the overall operation of the system 100. Thesystem controller 120 may be configured and programmed to control dataand/or power aspects of the various components. Although the systemcontroller 120 is represented as a single structure in FIG. 1, it isunderstood that the system controller 120 may include multiple separatecomponents (e.g., processors) that are distributed throughout the system100 at different locations. In some implementations, one or morecomponents may be integrated with the base instrument 102 and one ormore components may be located remotely with respect to the baseinstrument 102.

IV. Flow Cell

FIGS. 3-4 depict an example of a flow cell 400 that may be used withsystem 100. Flow cell of this example includes a body defining aplurality of elongate flow channels 410, which are recessed below anupper surface 404 of the body 402. The flow channels 410 are generallyparallel with each other and extend along substantially the entirelength of body 402. While five flow channels 410 are shown, a flow cell400 may include any other suitable number of flow channels 410,including more or fewer than five flow channels 410. The flow cell 400of this example also includes a set of inlet ports 420 and a set ofoutlet ports 422, with each port 420, 422 being associated with acorresponding flow channel 410. Thus, each inlet port 420 may beutilized to communicate fluids (e.g., reagents, etc.) to thecorresponding channel 410; while each outlet port 422 may be utilized tocommunicate fluids from the corresponding flow channel 410.

In some versions, the flow cell 400 is directly integrated into the flowcell receiving portion 220 of the removable cartridge 200. In some otherversions, the flow cell 400 is removably coupled with the flow cellreceiving portion 220 of the removable cartridge 200. In versions wherethe flow cell 400 is either directly integrated into the flow cellreceiving portion 220 or removably coupled with the flow cell receivingportion 220, the flow channels 410 of the flow cell 400 may receivefluids from the consumable reagent portion 210 via the inlet ports 420,which may be fluidly coupled with reagents stored in the consumablereagent portion 210. Of course, the flow channels 410 may be coupledwith various other fluid sources or reservoirs, etc., via the ports 420,422. As another illustrative variation, some versions of consumablecartridge 300 may be configured to removably receive or otherwiseintegrate the flow cell 400. In such versions, the flow channels 410 ofthe flow cell 400 may receive fluids from the reagent chambers 310, 320,330 via the inlet ports 420. Other suitable ways in which the flow cell400 may be incorporated into the system 100 will be apparent to thoseskilled in the art in view of the teachings herein.

FIG. 4 shows a flow channel 410 of the flow cell 400 in greater detail.As shown, the flow channel 410 includes a plurality of wells 430 formedin a base surface 412 of the flow channel 410. As will be described ingreater detail below each well 430 is configured to contain DNA strandsor other polynucleotides, such as machine-written polynucleotides. Insome versions, each well 430 has a cylindraceous configuration, with agenerally circular cross-sectional profile. In some other versions, eachwell 430 has a polygonal (e.g., hexagonal, octagonal, etc.)cross-sectional profile. Alternatively, wells 430 may have any othersuitable configuration. It should also be understood that wells 430 maybe arranged in any suitable pattern, including but not limited to a gridpattern.

FIG. 5 shows a portion of a channel within a flow cell 500 that is anexample of a variation of the flow cell 400. In other words, the channeldepicted in FIG. 5 is a variation of the flow channel 410 of the flowcell 400. This flow cell 500 is operable to read polynucleotide strands550 that are secured to the floor 534 of wells 530 in the flow cell 500.By way of example only, the floor 534 where polynucleotide strands 550are secured may include a co-block polymer capped with azido. By way offurther example only, such a polymer may comprise apoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM)coating provided in accordance with at least some of the teachings ofU.S. Pat. No. 9,012,022, entitled “Polymer Coatings,” issued Apr. 21,2015, which is incorporated by reference herein in its entirety. Such apolymer may be incorporated into any of the various flow cells describedherein.

In the present example, the wells 530 are separated by interstitialspaces 514 provided by the base surface 512 of the flow cell 500. Eachwell 530 has a sidewall 532 and a floor 534. The flow cell 500 in thisexample is operable to provide an image sensor 540 under each well 530.In some versions, each well 530 has at least one corresponding imagesensor 540, with the image sensors 540 being fixed in position relativeto the wells 530. Each image sensor 540 may comprise a CMOS imagesensor, a CCD image sensor, or any other suitable kind of image sensor.By way of example only, each well 530 may have one associated imagesensor 540 or a plurality of associated image sensors 540. As anothervariation, a single image sensor 540 may be associated with two or morewells 530. In some versions, one or more image sensors 540 move relativeto the wells 530, such that a single image sensor 540 or single group ofimage sensors 540 may be moved relative to the wells 530. As yet anothervariation, the flow cell 500 may be movable in relation to the singleimage sensor 540 or single group of image sensors 540, which may be atleast substantially fixed in position.

Each image sensor 540 may be directly incorporated into the flow cell500. Alternatively, each image sensor 540 may be directly incorporatedinto a cartridge such as the removable cartridge 200, with the flow cell500 being integrated into or otherwise coupled with the cartridge. Asyet another illustrative variation, each image sensor 540 may bedirectly incorporated into the base instrument 102 (e.g., as part of thedetection assembly 110 noted above). Regardless of where the imagesensor(s) 540 is/are located, the image sensor(s) 540 may be integratedinto a printed circuit that includes other components (e.g., controlcircuitry, etc.). In versions where the one or more image sensors 540are not directly incorporated into the flow cell 500, the flow cell 500may include optically transmissive features (e.g., windows, etc.) thatallow the one or more image sensors 540 to capture fluorescence emittedby the one or more fluorophores associated with the polynucleotidestrands 550 that are secured to the floors 534 of the wells 530 in theflow cell 500 as described in greater detail below. It should also beunderstood that various kinds of optical elements (e.g., lenses, opticalwaveguides, etc.) may be interposed between the floors 534 of the wells530 and the corresponding image sensor(s) 540.

As also shown in FIG. 5, a light source 560 is operable to project light562 into the well 530. In some versions, each well 530 has at least onecorresponding light source 560, with the light sources 560 being fixedin position relative to the wells 530. By way of example only, each well530 may have one associated light source 560 or a plurality ofassociated light sources 560. As another variation, a single lightsource 560 may be associated with two or more wells 530. In some otherversions, one or more light sources 560 move relative to the wells 530,such that a single light source 560 or single group of light sources 560may be moved relative to the wells 530. As yet another variation, theflow cell 500 may be movable in relation to the single light source 560or single group of light sources 560, which may be substantially fixedin position. By way of example only, each light source 560 may includeone or more lasers. In another example, the light source 560 may includeone or more diodes.

Each light source 560 may be directly incorporated into the flow cell500. Alternatively, each light source 560 may be directly incorporatedinto a cartridge such as the removable cartridge 200, with the flow cell500 being integrated into or otherwise coupled with the cartridge. Asyet another illustrative variation, each light source 560 may bedirectly incorporated into the base instrument 102 (e.g., as part of thedetection assembly 110 noted above). In versions where the one or morelight sources 560 are not directly incorporated into the flow cell 500,the flow cell 500 may include optically transmissive features (e.g.,windows, etc.) that allow the wells 530 to receive the light emitted bythe one or more light source 560, to thereby enable the light to reachthe polynucleotide strands 550 that are secured to the floor 534 of thewells 530. It should also be understood that various kinds of opticalelements (e.g., lenses, optical waveguides, etc.) may be interposedbetween the wells 530 and the corresponding light source(s) 560.

As described elsewhere herein and as is shown in block 590 of FIG. 6, aDNA reading process may begin with performing a sequencing reaction inthe targeted well(s) 530 (e.g., in accordance with at least some of theteachings of U.S. Pat. No. 9,453,258, entitled “Methods and Compositionsfor Nucleic Acid Sequencing,” issued Sep. 27, 2016, which isincorporated by reference herein in its entirety). Next, as shown inblock 592 of FIG. 6, the light source(s) 560 is/are activated over thetargeted well(s) 530 to thereby illuminate the targeted well(s) 530. Theprojected light 562 may cause a fluorophore associated with thepolynucleotide strands 550 to fluoresce. Accordingly, as shown in block594 of FIG. 6, the corresponding image sensor(s) 540 may detect thefluorescence emitted from the one or more fluorophores associated withthe polynucleotide strands 550. The system controller 120 of the baseinstrument 102 may drive the light source(s) 560 to emit the light. Thesystem controller 120 of the base instrument 102 may also process theimage data obtained from the image sensor(s) 540, representing thefluorescent emission profiles from the polynucleotide strands 550 in thewells 530. Using this image data from the image sensor(s) 540, and asshown in block 596 of FIG. 6, the system controller 120 may determinethe sequence of bases in each polynucleotide strand 550. By way ofexample only, this process and equipment may be utilized to map a genomeor otherwise determine biological information associated with anaturally occurring organism, where DNA strands or other polynucleotidesare obtained from or otherwise based on a naturally occurring organism.Alternatively, the above-described process and equipment may be utilizedto obtain data stored in machine-written DNA as will be described ingreater detail below.

By way of further example only, when carrying out the above-describedprocedure shown in FIG. 6, time space sequencing reactions may utilizeone or more chemistries and imaging events or steps to differentiatebetween a plurality of analytes (e.g., four nucleotides) that areincorporated into a growing nucleic acid strand during a sequencingreaction; or alternatively, fewer than four different colors may bedetected in a mixture having four different nucleotides while stillresulting in the determination of the four different nucleotides (e.g.,in a sequencing reaction). A pair of nucleotide types may be detected atthe same wavelength, but distinguished based on a difference inintensity for one member of the pair compared to the other, or based ona change to one member of the pair (e.g., via chemical modification,photochemical modification, or physical modification) that causesapparent signal to appear or disappear compared to the signal detectedfor the other member of the pair.

V. Machine-Writing Biological Material

In some implementations, a system 100 such as the system 100 shown inFIG. 1 may be configured to synthesize biological materials (e.g.polynucleotide, such as DNA) to encode data that may later be retrievedthrough the performance of assays such as those described above. In someimplementations, this type of encoding may be performed by assigningvalues to nucleotide bases (e.g., binary values, such as 0 or 1, ternaryvalues such as 0, 1 or 2, etc.), converting the data to be encoded intoa string of the relevant values (e.g., converting a textual message intoa binary string using the ASCII encoding scheme), and then creating oneor more polynucleotides made up of nucleotides having bases in asequence corresponding to the string obtained by converting the data.

In some implementations, the creation of such polynucleotides may beperformed using a version of the flow cell 400 having an array of wells630 that are configured as shown in FIG. 7. FIG. 7 shows a portion of achannel within a flow cell 600 that is an example of a variation of theflow cell 400. In other words, the channel depicted in FIG. 7 is avariation of the flow channel 410 of the flow cell 400. In this example,each well 630 is recessed below a base surface 612 of the flow cell 600.The wells 630 are thus spaced apart from each other by interstitialspaces 614. By way of example only, the wells 630 may be arranged in agrid or any other suitable pattern along the base surface 612 of theflow cell 600. Each well 630 of this example includes a sidewall 632 anda floor 634. Each well 630 of this example further includes a respectiveelectrode assembly 640 positioned on the floor 634 of the well 630. Insome versions, each electrode assembly 640 includes just a singleelectrode element. In some other versions, each electrode assembly 640includes a plurality of electrode elements or segments. The terms“electrode” and “electrode assembly” should be read herein as beinginterchangeable.

Base instrument 102 is operable to independently activate electrodeassemblies 640, such that one or more electrode assemblies 640 may be inan activated state while one or more other electrode assemblies 640 arenot in an activated state. In some versions, a CMOS device or otherdevice is used to control electrode assemblies 640. Such a CMOS devicemay be integrated directly into the flow cell 600, may be integratedinto a cartridge (e.g., cartridge 200) in which the flow cell 600 isincorporated, or may be integrated directly into the base instrument102. As shown in FIG. 7, each electrode assembly 640 extends along thefull width of floor 634, terminating at the sidewall 632 of thecorresponding well 630. In other versions, each electrode assembly 640may extend along only a portion of the floor 634. For instance, someversions of electrode assembly 640 may terminate interiorly relative tothe sidewall 632. While each electrode assembly 540 is schematicallydepicted as a single element in FIG. 5, it should be understood thateach electrode assembly 540 may in fact be formed by a plurality ofdiscrete electrodes rather than just consisting of one single electrode.

As shown in FIG. 7, specific polynucleotide strands 650 may be createdin individual wells 630 by activating the electrode assembly 640 of therelevant wells 630 to electrochemically generate acid that may deprotectthe end group of the polynucleotide strand 650 in the well 630. By wayof example only, polynucleotide strands 650 may be chemically attachedto the surface at the bottom of the well 630 using linkers havingchemistries such as silane chemistry on one end and DNA synthesiscompatible chemistry (e.g., a short oligo for enzyme to bind to) on theother end.

To facilitate reagent exchange (e.g., transmission of a deblockingagent), each electrode assembly 640 and the floor 634 of each well 630may include at least one opening 660 in this example. The openings 660may be fluidly coupled with a flow channel 662 that extends underneaththe wells 630, below the floors 634. To provide such an opening 660through the electrode assembly 640, the electrode assembly 640 may beannular in shape, may be placed in quadrants, may be placed on theperimeter or sidewall 632 of the well 630, or may be placed or shaped inother suitable manners to avoid interference with reagent exchangeand/or passage of light (e.g., as may be used in a sequencing processthat involved detection of fluorescent emissions). In otherimplementations, reagents may be provided into the flow channel of theflow cell 600 without the openings 660. It should be understood that theopenings 660 may be optional and may be omitted in some versions.Similarly, the flow channel 662 may be optional and may be omitted insome versions.

FIG. 9 shows an example of a form that electrode assembly 640 may take.In this example, electrode assembly 640 includes four discrete electrodesegments 642, 644, 646, 648 that together define an annular shape. Theelectrode segments 642, 644, 646, 648 are thus configured as discreteyet adjacent quadrants of a ring. Each electrode segment 642, 644, 646,648 may be configured to provide a predetermined charge that is uniquelyassociated with a particular nucleotide. For instance, electrode segment642 may be configured to provide a charge that is uniquely associatedwith adenine; electrode segment 644 may be configured to provide acharge that is uniquely associated with cytosine; electrode segment 646may be configured to provide a charge that is uniquely associated withguanine; and electrode segment 648 may be configured to provide a chargethat is uniquely associated with thymine. When a mixture of those fournucleotides are flowed through the flow channel above the wells 630,activation of electrode segments 642, 644, 646, 648 may cause thecorresponding nucleotides from that flow to adhere to the strand 650.Thus, when electrode segment 642 is activated, it may effect writing ofadenine to the strand 650; when electrode segment 644 is activated, itmay effect writing of cytosine to the strand 650; when electrode segment646 is activated, it may effect writing of guanine to the strand 650;and when electrode segment 648 is activated, it may effect writing ofthymine to the strand 650. This writing may be provided by the activatedelectrode segment 642, 644, 646, 648 hybridizing the inhibitor of theenzyme for the pixel associated with the activated electrode segment642, 644, 646, 648. While electrode segments 642, 644, 646, 648 areshown as forming an annular shape in FIG. 9, it should be understoodthat any other suitable shape or shapes may be formed by electrodesegments 642, 644, 646, 648. In still other implementations, a singleelectrode may be utilized for the electrode assembly 640 and the chargemay be modulated to incorporate various nucleotides to be written to theDNA strand or other polynucleotide.

As another example, the electrode assembly 640 may be activated toprovide a localized (e.g., localized within the well 630 in which theelectrode assembly 640 is disposed), electrochemically generated changein pH; and/or electrochemically generate a moiety (e.g., a reducing oroxidizing reagent) locally to remove a block from a nucleotide. As yetanother variation, different nucleotides may have different blocks; andthose blocks may be photocleaved based on a wavelength of lightcommunicated to the well 630 (e.g., light 562 projected from the lightsource 560). As still another variation, different nucleotides may havedifferent blocks; and those blocks may be cleaved based on certain otherconditions. For instance, one of the four blocks may be removed based ona combination of a reducing condition plus either high local pH or lowlocal pH; another of the four blocks may be removed based on acombination of an oxidative condition plus either high local pH or lowlocal pH; another of the four blocks may be removed based on acombination of light and a high local pH; and another of the four blocksmay be removed based on a combination of light and a low local pH. Thus,four nucleotides may be incorporated at the same time, but withselective unblocking occurring in response to four different sets ofconditions.

The electrode assembly 640 further defines the opening 660 at the centerof the arrangement of the electrode segments 642, 644, 646, 648. Asnoted above, this opening 660 may provide a path for fluid communicationbetween the flow channel 662 and the wells 630, thereby allowingreagents, etc. that are flowed through the flow channel 662 to reach thewells 630. As also noted above, some variations may omit the flowchannel 662 and provide communication of reagents, etc. to the wells 630in some other fashion (e.g., through passive diffusion, etc.).Regardless of whether fluid is communicated through the opening 660, theopening 660 may provide a path for optical transmission through thebottom of the well 630 during a read cycle, as described herein. In someversions, the opening 660 may be optional and may thus be omitted. Inversions where the opening 660 is omitted, fluids may be communicated tothe wells 630 via one or more flow channels that are above the wells 630or otherwise positioned in relation to the wells 630. Moreover, theopening 660 may not be needed for providing a path for opticaltransmission through the bottom of the well 630 during a read cycle. Forinstance, as described below in relation to the flow cell 601, theelectrode assembly 640 may comprise an optically transparent material(e.g., optically transparent conducting film (TCF), etc.), and the flowcell 600 itself may comprise an optically transparent material (e.g.,glass), such that the electrode assembly 640 and the material formingthe flow cell 600 may allow the fluorescence emitted from the one ormore fluorophores associated with the machine-written polynucleotidestrands 650 to reach an image sensor 540 that is under the well 630.

FIG. 8 shows an example of a process that may be utilized in the flowcell 600 to machine-write polynucleotides or other nucleotide sequences.At the beginning of the process, as shown in the first block 690 of FIG.8, nucleotides may be flowed into the flow cell 600, over the wells 630.As shown in the next block 692 in FIG. 8, the electrode assembly 640 maythen be activated to write a first nucleotide to a primer at the bottomof a targeted well 630. As shown in the next block 694 of FIG. 8, aterminator may then be cleaved off the first nucleotide that was justwritten in the targeted well 630. Various suitable ways in which aterminator may be cleaved off the first nucleotide will be apparent tothose skilled in the art in view of the teachings herein. Once theterminator is cleaved off the first nucleotide, as shown in the nextblock 696 of FIG. 8, the electrode assembly 640 may be activated towrite a second nucleotide to the first nucleotide. While not shown inFIG. 8, a terminator may be cleaved off the second nucleotide, then athird nucleotide may be written to the second nucleotide, and so onuntil the desired sequence of nucleotides has been written.

In some implementations, encoding of data via synthesis of biologicalmaterials such as DNA may be performed in other manners. For example, insome implementations, the flow cell 600 may lack the electrode assembly640 altogether. For instance, deblock reagents may be selectivelycommunicated from the flow channel 662 to the wells 630 through theopenings 660. This may eliminate the need for electrode assemblies 640to selectively activate nucleotides. As another example, an array ofwells 630 may be exposed to a solution containing all nucleotide basesthat may be used in encoding the data, and then individual nucleotidesmay be selectively activated for individual wells 630 by using lightfrom a spatial light modulator (SLM). As another example, in someimplementations individual bases may be assigned combined values (e.g.,adenine may be used to encode the binary couplet 00, guanine may be usedto encode the binary couplet 01, cytosine may be used to encode thebinary couplet 10, and thymine may be used to encode the binary couplet11) to increase the storage density of the polynucleotides beingcreated. Other examples are also possible and will be immediatelyapparent to those skilled in the art in light of this disclosure.Accordingly, the above description of synthesizing biological materialssuch as DNA to encode data should be understood as being illustrativeonly; and should not be treated as limiting.

VI. Reading Machine-Written Biological Material

After polynucleotide strands 650 have been machine-written in one ormore wells 630 of a flow cell 600, the polynucleotide strands 650 may besubsequently read to extract whatever data or other information wasstored in the machine-written polynucleotide strands 650. Such a readingprocess may be carried out using an arrangement such as that shown inFIG. 5 and described above. In other words, one or more light sources560 may be used to illuminate one or more fluorophores associated withthe machine-written polynucleotide strands 650; and one or more imagesensors 540 may be used to detect the fluorescent light emitted by theilluminated one or more fluorophores associated with the machine-writtenpolynucleotide strands 650. The fluorescence profile of the lightemitted by the illuminated one or more fluorophores associated with themachine-written polynucleotide strands 650 may be processed to determinethe sequence of bases in the machine-written polynucleotide strands 650.This determined sequence of bases in the machine-written polynucleotidestrands 650 may be processed to determine the data or other informationthat was stored in the machine-written polynucleotide strands 650.

In some versions, the machine-written polynucleotide strands 650 remainin the flow cell 600 containing wells 630 for a storage period. When itis desired to read the machine-written polynucleotide strands 650, theflow cell 600 may permit the machine-written polynucleotide strands 650to be read directly from the flow cell. By way of example only, the flowcell 600 containing wells 630 may be received in a cartridge (e.g.,cartridge 200) or base instrument 102 containing light sources 560and/or image sensors 540, such that the machine-written polynucleotidestrands 650 are read directly from the wells 630.

As another illustrative example, the flow cell containing wells 630 maydirectly incorporate one or both of light source(s) 560 or imagesensor(s) 540. FIG. 10 shows an example of a flow cell 601 that includeswells 630 with electrode assemblies 640, one or more image sensors 540,and a control circuit 670. Like in the flow cell 500 depicted in FIG. 5,the flow cell 601 of this example is operable to receive light 562projected from a light source 560. This projected light 562 may causeone or more fluorophores associated with the machine-writtenpolynucleotide strands 650 to fluoresce; and the corresponding imagesensor(s) 540 may capture the fluorescence emitted from the one or morefluorophores associated with the machine-written polynucleotide strands650.

As noted above in the context of the flow cell 500, each well 650 of theflow cell 601 may include its own image sensor 540 and/or its own lightsource 560; or these components may be otherwise configured and arrangedas described above. In the present example, the fluorescence emittedfrom the one or more fluorophores associated with the machine-writtenpolynucleotide strands 650 may reach the image sensor 540 via theopening 660. In addition, or in the alternative, the electrode assembly640 may comprise an optically transparent material (e.g., opticallytransparent conducting film (TCF), etc.), and the flow cell 601 itselfmay comprise an optically transparent material (e.g., glass), such thatthe electrode assembly 640 and the material forming the flow cell 601may allow the fluorescence emitted from the one or more fluorophoresassociated with machine-written polynucleotide strands 650 to reach theimage sensor 540. Moreover, various kinds of optical elements (e.g.,lenses, optical waveguides, etc.) may be interposed between the wells650 and the corresponding image sensor(s) to ensure that the imagesensor 540 is only receiving fluorescence emitted from the one or morefluorophores associated with the machine-written polynucleotide strands650 of the desired well(s) 630.

In the present example, the control circuit 670 is integrated directlyinto the flow cell 601. By way of example only, the control circuit 670may comprise a CMOS chip and/or other printed circuitconfigurations/components. The control circuit 670 may be incommunication with the image sensor(s) 540, the electrode assembly(ies)640, and/or the light source 560. In this context, “in communication”means that the control circuit 670 is in electrical communication withimage sensor(s) 540, the electrode assembly(ies) 640, and/or the lightsource 560. For instance, the control circuit 670 may be operable toreceive and process signals from the image sensor(s) 540, with thesignals representing images that are picked up by the image sensor(s)540. “In communication” in this context may also include the controlcircuit 670 providing electrical power to the image sensor(s) 540, theelectrode assembly(ies) 640, and/or the light source 560.

In some versions, each image sensor 540 has a corresponding controlcircuit 670. In some other versions, a control circuit 670 is coupledwith several, if not all, of the image sensors in the flow cell 601.Various suitable components and configurations that may be used toachieve this will be apparent to those skilled in the art in view of theteachings herein. It should also be understood that the control circuit670 may be integrated, in whole or in part, in a cartridge (e.g.,removable cartridge 200) and/or in the base instrument 102, in additionto or in lieu of being integrated into the flow cell 601.

As still another illustrative example, regardless of whether awrite-only flow cell like the flow cell 600 of FIG. 7 or a read-writeflow cell like the flow cell 601 of FIG. 10 is used, the machine-writtenpolynucleotide strands 650 may be transferred from wells 630 after beingsynthesized. This may occur shortly after the synthesis is complete,right before the machine-written polynucleotide strands 650 are to beread, or at any other suitable time. In such versions, themachine-written polynucleotide strands 650 may be transferred to aread-only flow cell like the flow cell 500 depicted in FIG. 5; and thenbe read in that read-only flow cell 500. Alternatively, any othersuitable devices or processes may be used.

In some implementations, reading data encoded through the synthesis ofbiological materials may be achieved by determining the well(s) 630storing the synthesized strand(s) 650 of interest and then sequencingthose strands 650 using techniques such as those described previously(e.g., sequencing-by-synthesis). In some implementations, to facilitatereading data stored in nucleotide sequences, when data is stored, anindex may be updated with information showing the well(s) 630 where thestrand(s) 650 encoding that data was/were synthesized. For example, whenan implementation of a system 100 configured to synthesize strands 650capable of storing up to 256 bits of data is used to store a one megabit(1,048,576 bit) file, the system controller 120 may perform steps suchas: 1) break the file into 4,096 256 bit segments; 2) identify asequence of 4,096 wells 630 in the flow cell 600, 601 that were notcurrently being used to store data; 3) write the 4,096 segments to the4,096 wells 430, 530; 4) update an index to indicate that the sequencestarting with the first identified well 630 and ending at the lastidentified well 630 was being used to store the file. Subsequently, whena request to read the file was made, the index may be used to identifythe well(s) 630 containing the relevant strand(s) 650, the strand(s) 650from those wells 630 may be sequenced, and the sequences may be combinedand converted into the appropriate encoding format (e.g., binary), andthat combined and converted data may then be returned as a response tothe read request.

In some implementations, reading of data previously encoded viasynthesis of biological materials may be performed in other manners. Forexample, in some implementations, if a file corresponding to 4,096 wells630 was to be written, rather than identifying 4,096 sequential wells630 to write it to, a controller may identify 4,096 wells 630 and thenupdate the index with multiple locations corresponding to the file inthe event that those wells 630 did not form a continuous sequence. Asanother example, in some implementations, rather than identifyingindividual wells 630, a system controller 120 may group wells 630together (e.g., into groups of 128 wells 630), thereby reducing theoverhead associated with storing location data (i.e., by reducing theaddressing requirements from one address per well 630 to one address pergroup of wells 630). As another example, in implementations that storedata reflecting the location of wells 630 where DNA strands or otherpolynucleotides have been synthesized, that data may be stored invarious ways, such as sequence identifiers (e.g., well 1, well 2, well3, etc.) or coordinates (e.g., X and Y coordinates of a well's locationin an array).

As another example, in some implementations, rather than reading strands650 from the wells 630 in which they were synthesized, strands 650 maybe read from other locations. For instance, strands 650 may besynthesized to include addresses, and then cleaved from the wells 630and stored in a tube for later retrieval, during which the includedaddress information may be used to identify the strands 650corresponding to particular files. As another illustrative example, thestrands 650 may be copied off the surface using polymerase and theneluted & stored in tube. Alternatively, the strands 650 may be copied onto a bead using biotinylated oligos hybridized to DNA strands or otherpolynucleotides and capturing extended products on streptavidin beadsthat are dispensed in the wells 630. Other examples are also possibleand will be immediately apparent to those of skill in the art in lightof this disclosure. Accordingly, the above description of retrievingdata encoded through the synthesis of biological materials should beunderstood as being illustrative only; and should not be treated aslimiting.

Implementations described herein may utilize a polymer coating for asurface of a flow cell, such as that described in U.S. Pat. No.9,012,022, entitled “Polymer Coatings,” issued Apr. 21, 2015, which isincorporated by reference herein in its entirety. Implementationsdescribed herein may utilize one or more labelled nucleotides having adetectable label and a cleavable linker, such as those described in U.S.Pat. No. 7,414,116, entitled “Labelled Nucleotide Strands,” issued Aug.19, 2008, which is incorporated by reference herein in its entirety. Forinstance, implementations described herein may utilize a cleavablelinker that is cleavable with by contact with water-soluble phosphinesor water-soluble transition metal-containing catalysts having afluorophore as a detectable label. Implementations described herein maydetect nucleotides of a polynucleotide using a two-channel detectionmethod, such as that described in U.S. Pat. No. 9,453,258, entitled“Methods and Compositions for Nucleic Acid Sequencing,” issued Sep. 27,2016, which is incorporated by reference herein in its entirety. Forinstance, implementations described herein may utilize afluorescent-based SBS method having a first nucleotide type detected ina first channel (e.g., dATP having a label that is detected in the firstchannel when excited by a first excitation wavelength), a secondnucleotide type detected in a second channel (e.g., dCTP having a labelthat is detected in a second channel when excited by a second excitationwavelength), a third nucleotide type detected in both the first andsecond channel (e.g., dTTP having at least one label that is detected inboth channels when excited by the first and/or second excitationwavelength), and a fourth nucleotide type that lacks a label that isnot, or that is minimally, detected in either channel (e.g., dGTP havingno label). Implementations of the cartridges and/or flow cells describedherein may be constructed in accordance with one or more teachingsdescribed in U.S. Pat. No. 8,906,320, entitled “Biosensors forBiological or Chemical Analysis and Systems and Methods for Same,”issued Dec. 9, 2014, which is incorporated by reference herein in itsentirety; U.S. Pat. No. 9,512,422, entitled “Gel Patterned Surfaces,”issued Dec. 6, 2016, which is incorporated by reference herein in itsentirety; U.S. Pat. No. 10,254,225, entitled “Biosensors for Biologicalor Chemical Analysis and Methods of Manufacturing the Same,” issued Apr.9, 2019, which is incorporated by reference herein in its entirety;and/or U.S. Pub. No. 2018/0117587, entitled “Cartridge Assembly,”published May 3, 2018, which is incorporated by reference herein in itsentirety.

VII. Features to Contain Reactions Within Wells and Prevent DiffusionBetween Wells

In a DNA storage device that provides reading and writing capabilityamong various wells 630 of a flow cell 601, it may be beneficial tocontain reactions to the wells 630 in which the reactions are intendedto occur. In some instances, there may be a risk that the reactionwithin one well 630 may not be fully contained within that well 630.This may occur during a writing process (e.g., in the case of well 630)or during a reading process (e.g., in the case of well 530, 630).Similarly, it may be beneficial to prevent inter-well diffusion, tothereby prevent the occurrence of chemical cross-talk between adjacentwells 630 in a flow cell 601. Each individual well 601 may incorporatefeatures (e.g., added depth, other features as described below, etc.)that provide such containment and prevent such diffusion. In addition,or in the alternative, the flow cell 601 may include features in thespaces 614 between wells 630 to provide such containment and preventsuch diffusion. Several illustrative examples of containment anddiffusion prevention features are described in greater detail below.While the following examples are provided separately with reference toseparate drawings, it should be understood that the features of thefollowing examples may be combined in numerous ways in the same flowcell. Thus, the containment and diffusion prevention features describedbelow should not be viewed as being exclusive of each other.

A. Flow Cell with Valves in Wells

FIGS. 11A-11B show an example of a flow cell 700 that may be used towrite DNA strands (or other polynucleotides) as described herein. Exceptas otherwise described below, the flow cell 700 of this example may beconfigured and operable like the other flow cells 400, 500, 600, 601described herein. For instance, while an image sensor 540 and a controlcircuit 670 are not shown in FIGS. 11A-11B, variations of flow cell 700may in fact include an image sensor 540 and/or a control circuit 670like the flow cell 601 of FIG. 10. Similarly, the flow cell 700 of thisexample may incorporate or otherwise be placed under a light source 560like the flow cell 601 of FIG. 10.

The flow cell 700 of the present example includes a plurality of wells730 that are recessed below a base surface 712. While only one well 730is shown in FIGS. 11A-11B, it should be understood that the flow cell700 may include several wells 730 that are configured just like thedepicted well 730; and that the wells 730 may be arranged in anysuitable pattern. The flow cell 700 of this example further defines anupper flow channel 764 (e.g., corresponding to the channels 410 shown inFIGS. 3-4), with wells 730 being positioned to receive fluid that isflowed through upper flow channel 764. The flow cell 700 also defines alower flow channel 762 that is positioned underneath the wells 730. Inthe present example, the two flow channels 762, 764 are fluidicallycoupled with different fluid sources. By way of example only, the upperflow channel 764 may be fluidically coupled with one or more fluidsources containing nucleotides; while the lower flow channel 762 may befluidically coupled with one or more fluid sources containing deblockingagents. Other suitable kinds of fluids, and contents of such fluids,that may be communicated through the respective flow channels 762, 764will be apparent to those skilled in the art in view of the teachingsherein.

Each well 730 of this example includes a sidewall 732 and a floor 734.Each well 730 of this example further includes a respective electrodeassembly 740 positioned on the floor 734 of the well 730. In someversions, each electrode assembly 740 includes just a single electrodeelement. In some other versions, each electrode assembly 740 includes aplurality of electrode elements or segments (e.g., like the electrodesegments 642, 644, 646, 648 described above, etc.).

Unlike the flow cell 601 shown in FIG. 10, the flow cell 700 of thisexample includes a valve 780 that is positioned in an opening 760extending from the floor 734 of the well 730 to the lower flow channel762. The valve 780 is in communication with a controller 790 that isoperable to drive the valve 780 to transition between a closed state(FIG. 11A) and an open state (FIG. 11B). In some versions, thecontroller 790 is in electrical communication with the valve 780, suchthat the controller 790 is operable to provide electrical signals to thevalve 780 to thereby activate the valve to thereby transition the valve780 between open and closed states. When the valve 780 is in the closedstate (FIG. 11A), the orifice 782 of the valve is reduced to a pointwhere fluid cannot flow through the orifice 782, such that fluid thatflows through the lower flow channel 762 cannot reach the well 730. Whenthe valve 780 is in the open state (FIG. 11B), the orifice 782 of thevalve is sized to permit fluid to flow through the orifice 782, suchthat fluid that flows through the lower flow channel 762 may flow intothe well 730.

In some instances, components or techniques are used to provide apressure differential between the well 730 and the lower flow channel762 to promote the flow of fluid from the lower flow channel 762 intothe well 730 when the valve 780 is in the open state. By way of exampleonly, a pump (not shown), such as an electrokinetic pump or other kindof pump, may be utilized to promote the flow of fluid from the lowerflow channel 762 into the well 730 when the valve 780 is in the openstate. As another illustrative example, the lower flow channel 762 mayinclude valves or other flow restriction devices between adjacentopenings 760 of wells 730 to selectively restrict flow through the lowerflow channel 762. For instance, when a valve 780 in a particular opening760 is in activated to achieve an open state, another valve (not shown)that is downstream of that opening 760 in the lower flow channel 762 maybe closed to provide a buildup of pressure in the lower flow channel762, thereby promoting the flow of fluid from the lower flow channelinto the corresponding well 730 via the opened valve 780 in the opening760 of that well 730. Other suitable components or techniques that maybe used to provide a pressure differential between the well 730 and thelower flow channel 762, or to otherwise promote the flow of fluid fromthe lower flow channel 762 into the well 730 when the valve 780 is inthe open state, will be apparent to those skilled in the art in view ofthe teachings herein.

The controller 790 may take a variety of forms and may drive the valve780 in numerous different ways, particularly depending on the nature ofthe valve 780. For instance, some versions of the valve 780 may comprisea hydrogel material that is configured to define a ring shape or hollowcylindraceous shape. In such versions, the hydrogel material maytransition between a swelled state or expanded state and a non-swelledstate or contracted state to transition the valve 780 between the closedstate (FIG. 11A) and the open state FIG. 11B, respectively. Thecontroller 790 may drive the hydrogel material to transition between theswelled state or expanded state and the non-swelled state or contractedstate by applying pH changes, reagents, and/or electrical flow. Varioussuitable ways in which the controller 790 may drive the hydrogelmaterial to transition between the swelled state and the non-swelledstate, and various components that may be incorporated into thecontroller or otherwise associate with the controller 790 to providesuch transitioning, will be apparent to those skilled in the art in viewof the teachings herein.

As another illustrative example, the valve 780 may comprise a heatswellable polymer. In such versions, the heat swellable polymer maytransition between a swelled state and a non-swelled state to transitionthe valve 780 between the closed state (FIG. 11A) and the open stateFIG. 11B, respectively. The controller 790 may drive the heat swellablepolymer to transition between the swelled state and the non-swelledstate by selectively heating the heat swellable polymer. For instance,the controller 790 may include one or more heating elements that are inthermal communication with the heat swellable polymer, such that thecontroller 790 may activate the heating elements to thereby cause theheat swellable polymer to swell, thereby causing the valve 780 to close.To open the valve 780, the controller 790 may deactivate the heatingelements, and the heat swellable polymer may then cool to the pointwhere the valve 780 reaches a cool state. In some instances, a resilientmaterial is incorporated into valve 780 to resiliently urge the valve780 to return to the open state when the heating is removed.

Some other versions of the valve 780 may comprise an electroactivepolymer. In such versions, the controller 790 may apply a voltage to thevalve 780 or remove the voltage from the valve 780 in order totransition the valve 780 between the open and closed states. Othersuitable forms that the valve 780 and the controller 790 may take willbe apparent to those skilled in the art in view of the teachings herein.While the controller 790 is shown as being positioned adjacent to thevalve 780 as a separate unit, such that each valve 780 has separatecontrollers 790, it should be understood that other versions may providean integrated circuit that provides control over all valves 780. Forinstance, a single CMOS chip in the flow cell 700 may be operable todrive all of the valves 780 in the flow cell 700. Even when thecontroller 790 is effectively embodied in a single device (e.g., asingle CMOS chip that controls all of the valves 780 in the flow cell700), such a unified controller 790 may still operate the valves 780independently of each other.

Regardless of what form the valve 780 takes, the various valves 780 inthe flow cell 700 may be selectively activated to control the flow offluids from the lower flow channel 762 to the corresponding well(s) 730.For instance, valves 780 may be selectively activated to control theflow of de-shielding/de-blocking agent from the lower flow channel 762to the corresponding well(s) 730. In other words, closed valves 780 willprevent selected wells 730 from receiving de-shielding/de-blocking agentfrom the lower flow channel 762, thereby enhancing control over whichwell 730 provides a reaction at a given moment. Since the valves 780 maybe independently operated in the present example, the valves 780 enablethe wells 730 to receive de-shielding/de-blocking agent from the lowerflow channel 762 independently or in any desired pattern.

B. Flow Cell with Pressure Gradient Feature

Another way in which a flow cell may provide containment within wells,and prevent diffusion between wells, is to provide a pressure gradientwithin each well. FIG. 12 shows one illustrative example of how this maybe carried out. In particular, FIG. 12 shows a flow cell 800 with a pumpassembly 810 having two ports 812, 814. Except for the inclusion of thepump assembly 810 and the ports 812, 814, the flow cell 800 of thisexample is configured and operable just like the flow cell 700 describedabove. Thus, like reference numerals indicate like components betweenthe two examples, and these overlapping components will not be furtherdescribed in the context of this flow cell 800. It should be understood,however, that the pump assembly 810 and the ports 812, 814 may beincorporated into flow cells of different configurations (e.g., a flowcell in which the valve 780 is omitted).

The pump assembly 810 may include an osmotic pump or any other suitablekind of pump. The ports 812, 814 include an upper port 812 and a lowerport 814. The ports 812, 814 open into the sidewall 732 of the well 730,such that the ports 812, 814 are fluidically coupled with the well 730.The upper port 812 is positioned near the top of the well 730 and thelower port 814 is positioned near the bottom of the well 730.

The pump assembly 810 is operable to provide a fluid flow or pressureprofile that varies across the depth of the well 730. For instance, thepump assembly 810 may provide a fluid flow or pressure profile where theflow or pressure is lower near the bottom of the well 730; with a flowor pressure that is higher near the top of the well 730. In some othervariations, the lower port 814 is omitted and the pump assembly 810draws fluid from the upper flow channel 764 and thereby provides ahigher flow or pressure near the top of the well 730 since the lowerregion of the well is not receiving a flow of fluid directly from thepump assembly 810. In either case, by providing this particular flow orpressure gradient within the well 730, the pump assembly 810 may assistin containing reactions within that well 730; and further preventdiffusion to adjacent wells 730.

The pump assembly 810 may be selectively activated by a controller thatis directly integrated into the flow cell 800. By way of example only,such an integrated controller may be incorporated into a CMOS chip. Insome such versions, the same CMOS chip or other controller also controlsother features of the flow cell 800 (e.g., the valve 780, the electrodeassembly 740, etc.). As another illustrative alternative, the pumpassembly 810 may be selectively activated by a controller that isdirectly integrated into a cartridge (e.g., the removable cartridge 200)that receives the flow cell 800. As still another illustrativealternative, the pump assembly 810 may be selectively activated by acontroller that is directly integrated into the base instrument 102.Moreover, components of the controller that selectively activates thepump assembly 810 may be distributed among two or more of the flow cell800, a cartridge that receives the flow cell 800, or the base instrument102. Various suitable components and arrangements that may be utilizedto provide control of the pump assembly 810 will be apparent to thoseskilled in the art in view of the teachings herein.

FIG. 13 shows a flow cell 900 with a bubble generator 910. Except forthe inclusion of the bubble generator 910, the flow cell 900 of thisexample is configured and operable just like the flow cell 700 describedabove. Thus, like reference numerals indicate like components betweenthe two examples, and these overlapping components will not be furtherdescribed in the context of this flow cell 900. It should be understood,however, that the bubble generator 910 may be incorporated into flowcells of different configurations (e.g., a flow cell in which the valve780 is omitted, a flow cell like the flow cell 800 of FIG. 12, etc.).

The bubble generator 910 of this example is positioned adjacent to thesidewall 732 of the well 730 and is operable to generate bubbles 910 inthe well 730. In the example shown in FIG. 13, the bubble generator 910is shown as being located in an intermediate position along the depth ofthe well 730. In other versions, the bubble generator 910 is locatednear the top of the well 730 or elsewhere. While just one bubblegenerator 910 is shown, it should be understood that each well 730 mayhave more than one associated bubble generator 910.

The bubbles 912 that are created by the bubble generator 910 may tend torise toward the top of the well 730. When the bubbles 912 are producedby the bubble generator 910 in an at least substantially constantstream, the bubbles 912 may effectively prevent liquid that is flowingthrough the upper flow channel 764 from reaching the well 730 in whichthe bubbles 912 are being generated. The bubbles 912 may also provide apressure gradient that effectively contains the reactions that areoccurring along the depth of the well 730 between the bubbles 912 andthe floor 734 of the well 730. As another illustrative example, thebubble generator 910 may generate a bubble dome over the well 730,thereby separating the well from the rest of the upper flow channel 764.In such versions, the bubble dome may prevent reactions from occurringwithin that well 730 as long as the bubble dome is present. In any ofthese variations, by generating bubbles 912, the bubble generator 910may assist in containing reactions within its associated well 730; andfurther prevent diffusion to adjacent wells 730.

The bubble generator 910 may be selectively activated by a controllerthat is directly integrated into the flow cell 900. By way of exampleonly, such an integrated controller may be incorporated into a CMOSchip. In some such versions, the same CMOS chip or other controller alsocontrols other features of the flow cell 900 (e.g., the valve 780, theelectrode assembly 740, etc.). As another illustrative alternative, thebubble generator 910 may be selectively activated by a controller thatis directly integrated into a cartridge (e.g., the removable cartridge200) that receives the flow cell 900. As still another illustrativealternative, the bubble generator 910 may be selectively activated by acontroller that is directly integrated into the base instrument 102.Moreover, components of the controller that selectively activates thebubble generator 910 may be distributed among two or more of the flowcell 900, a cartridge that receives the flow cell 900, or the baseinstrument 102. Various suitable components and arrangements that may beutilized to provide control of the bubble generator 910 will be apparentto those skilled in the art in view of the teachings herein.

In any of the foregoing examples, or in other variations of a flow cell800, 900, a pressure sensitive check valve may also be used to assist incontaining reactions within the wells 730 of a flow cell and/or preventdiffusion among wells 730 of the flow cell.

C. Flow Cell with Thermal Gradient Feature

Another way in which a flow cell may provide containment within wells,and prevent diffusion between wells, is to provide a thermal gradientwithin each well. Sharp thermal gradients may result in thermophoreticgradients of reagents within the well that effectively trap the reagentsnear an extreme condition (hot or cold). FIG. 14 shows one illustrativeexample of how thermal gradients may be provided within wells. Inparticular, FIG. 14 shows a flow cell 1000 with several thermal elements1010, 1012, 1020 integrated therein. Except for the inclusion of thethermal elements 1010, 1012, 1020, the flow cell 1000 of this example isconfigured and operable just like the flow cell 700 described above.Thus, like reference numerals indicate like components between the twoexamples, and these overlapping components will not be further describedin the context of this flow cell 1000. It should be understood, however,that the thermal elements 1010, 1012, 1020 may be incorporated into flowcells of different configurations (e.g., a flow cell in which the valve780 is omitted, a flow cell like the flow cell 800 of FIG. 12, a flowcell like the flow cell 900 of FIG. 13, etc.).

Each thermal element 1010, 1012, 1020 of the present example is operableto either increase or decrease the temperature of liquid that isadjacent to the thermal element 1010, 1012, 1020. The thermal elements1010 of this example are positioned along the sidewall 732 of the well730, near the bottom of the well 730. The thermal elements 1012 arepositioned along the sidewall 732 of the well 730, near the bottom ofthe well 730. The thermal elements 1020 are positioned along the basesurface 712 of the flow cell 1000, in the interstitial spaces 714between adjacent wells 730.

The thermal elements 1010 of this example comprise heating elements thatare operable to heat the liquid in the bottom region of the well 730.The thermal elements 1012 of this example comprise cryogenic elementsthat are operable to cool the liquid in the top region of the well 730.The thermal elements 1014 of this example comprise cryogenic elementsthat are operable to cool the liquid that is adjacent to theinterstitial spaces 714 between adjacent wells 730. With the thermalelements 1010 heating the lower region of the well 730, and with thethermal elements 1012 heating the lower region of the well 730, thethermal elements 1010, 1012 cooperate to provide a thermal gradientacross the depth of the well 730. This thermal gradient will helpcontain reactions within the well 730. With the thermal elements 1014cooling the interstitial spaces 714 between adjacent wells 730, thethermal elements will prevent diffusion from one well 730 to theadjacent well 730.

In some variations, thermal elements 1012 and/or thermal elements 1014are omitted. While not shown, each thermal element 1010, 1012, 1014 maybe selectively activated by a corresponding dedicated controller.Alternatively, a single controller (e.g., CMOS chip) may be utilized toselectively activate the thermal elements 1010, 1012, 1014. In someinstances, the entire set of thermal elements 1010, 1012 for a givenwell 730 is activated independently of the thermal elements 1010, 1012for other wells 730. As another variation, the thermal elements 1010,1012 for a given well 730 may be activated independently of each other.It should also be understood that the thermal elements 1014 may beactivated independently of the thermal elements 1010, 1012; or in tandemwith the thermal elements 1010, 1012.

The thermal elements 1010, 1012, 1014 may be selectively activated by acontroller that is directly integrated into the flow cell 1000. By wayof example only, such an integrated controller may be incorporated intoa CMOS chip. In some such versions, the same CMOS chip or othercontroller also controls other features of the flow cell 1000 (e.g., thevalve 780, the electrode assembly 740, etc.). As another illustrativealternative, the thermal elements 1010, 1012, 1014 may be selectivelyactivated by a controller that is directly integrated into a cartridge(e.g., the removable cartridge 200) that receives the flow cell 1000. Asstill another illustrative alternative, the thermal elements 1010, 1012,1014 may be selectively activated by a controller that is directlyintegrated into the base instrument 102. Moreover, components of thecontroller that selectively activates the thermal elements 1010, 1012,1014 may be distributed among two or more of the flow cell 1000, acartridge that receives the flow cell 1000, or the base instrument 102.Various suitable components and arrangements that may be utilized toprovide control of the thermal elements 1010, 1012, 1014 will beapparent to those skilled in the art in view of the teachings herein.

D. Flow Cell with pH Control Feature

Another way in which a flow cell may prevent diffusion between wells isto provide a pH gradient between adjacent wells. FIG. 15 shows oneillustrative example of how this may be carried out. In particular, FIG.15 shows a flow cell 1100 with a pH control feature 1110 integratedtherein. Except for the inclusion of the pH control feature 1110, theflow cell 1100 of this example is configured and operable just like theflow cell 700 described above. Thus, like reference numerals indicatelike components between the two examples, and these overlappingcomponents will not be further described in the context of this flowcell 1100. It should be understood, however, that the pH control feature1110 may be incorporated into flow cells of different configurations(e.g., a flow cell in which the valve 780 is omitted, a flow cell likethe flow cell 800 of FIG. 12, a flow cell like the flow cell 900 of FIG.13, a flow cell like the flow cell 1000 of FIG. 14, etc.).

The pH control feature 1110 of the present example is operable to adjustthe pH level within the well 730. By way of example only, the pH controlfeature may comprise a bubble generator, a set of electrodes, or someother feature. The flow cell 1100 may be configured to one or more pHcontrol features 1110 to provide pH levels that differ among differentwells 730. With the pH levels differing between the wells 730, theresulting pH gradient may prevent diffusion between adjacent wells 730.

The pH control feature 1110 may be selectively activated by a controllerthat is directly integrated into the flow cell 1100. By way of exampleonly, such an integrated controller may be incorporated into a CMOSchip. In some such versions, the same CMOS chip or other controller alsocontrols other features of the flow cell 1100 (e.g., the valve 780, theelectrode assembly 740, etc.). As another illustrative alternative, thepH control feature 1110 may be selectively activated by a controllerthat is directly integrated into a cartridge (e.g., the removablecartridge 200) that receives the flow cell 1100. As still anotherillustrative alternative, the pH control feature 1110 may be selectivelyactivated by a controller that is directly integrated into the baseinstrument 102. Moreover, components of the controller that selectivelyactivates the pH control feature 1110 may be distributed among two ormore of the flow cell 1100, a cartridge that receives the flow cell1100, or the base instrument 102. Various suitable components andarrangements that may be utilized to provide control of the pH controlfeature 1110 will be apparent to those skilled in the art in view of theteachings herein.

E. Flow Cell with Movable Barrier

FIGS. 16A-16B show an example of a flow cell 1200 with a barrier memberin the form of a droplet 1290 that is operable to selectively transitionbetween two different positions to thereby selectively transitionbetween providing a diffusion prevention barrier (FIG. 16A) andproviding a containment barrier (FIG. 16B). In particular, the flow cell1200 of this example employs digital microfluidics to provide controlledmovement of the droplet 1290. The flow cell 1200 of this exampleincludes a ceiling 1202, a lower flow channel 1262, an upper flowchannel 1264, and a plurality of wells 1230. The wells 1230 of thisexample have electrode assemblies 1240 (like other electrode assembliesdescribed herein); and openings 1260 providing a path for fluidcommunication between the lower flow channel 1262 and the correspondingwells 1230. While not shown in FIGS. 16A-16B, the openings 1260 mayinclude valves (e.g., like valves 780). Similarly, the flow cell 1200 ofthis example may include any of the other features of the other flowcells 400, 500, 600, 601, 700, 800, 900, 1000, 1100 described herein.

By way of example only, droplet 1290 may comprise oil or any othersuitable substance. While only one droplet 1290 is shown in FIGS.16A-16B, it should be understood that the flow cell 1200 of the presentexample includes a plurality of droplets 1290 positioned between anupper surface 1216 and a lower surface 1212 of the upper flow channel1264. The other droplets 1290 may be positioned in the flow cell 1200either in the interstitial spaces 1214 between wells 1230 (e.g., asshown in FIG. 16A) or over wells 1230 (e.g., as shown in FIG. 16B),depending on the activation state of electrodes in a correspondingelectrode layer 1284 as described below. An upper hydrophobic layer 1270is positioned along the ceiling 1202 of the flow cell 1200 and presentsthe upper surface 1216. A lower hydrophobic layer 1280 is positionedalong the bottom of the flow channel 1264 and presents the lower surface1212. The droplet 1290 is thus interposed between the hydrophobic layers1270, 1280.

In some variations, the droplet 1290 is positioned on the lower surface1212 without necessarily contacting the upper surface 1216. In suchvariations, the droplet 1290 may extend from the lower surface 1212toward the upper surface 1216, thereby restricting flow between adjacentwells 1230 without necessarily completely impeding flow between adjacentwells 1230.

In the present example, a dielectric layer 1282 is positioned under thelower hydrophobic layer 1280. An electrode layer 1284 is positionedunder the dielectric layer 1282. The electrode layer 1284 includes apatterned array of individually controllable electrode elements (notshown). The electrode elements are isolated from each other. Acontroller (not shown) is operable to selectively activate anddeactivate these electrode elements to provide movement of the droplet1290 between a first position as shown in FIG. 16A and a second positionas shown in FIG. 16B. In particular, the electrode elements selectivelyapply voltages to the droplets 1290 to provide controlled movement ofthe droplets 1290. The dielectric layer 1282 may provide build-up ofcharges and electrical field gradients to thereby provide movement ofthe droplets 1290. The droplets 1290 thus move based on electrowettingprinciples. While the droplet 1290 is shown as moving between a firstposition (FIG. 16A) and a second position (FIG. 16B) in this example, insome variations the droplet 1290 may expand and contract in response toapplied electrical field gradients. In some such versions, the droplet1290 may reside in an interstitial space 1214 when the droplet 1290 isin the contracted state (without covering any wells 1230); and cover oneor more wells 1230 when the droplet 1290 is in the expanded state.

When a droplet 1290 is positioned in the interstitial space 1214 betweenadjacent wells 1230 as shown in FIG. 16A, the droplet 1290 may serve asa physical barrier that prevents diffusion between adjacent wells 1230.When a droplet 1290 is positioned over a well 1230 as shown in FIG. 16B,the droplet 1290 may serve as a cap that physically contains reactionwithin the capped well 1230. By way of example only, a droplet 1290 maybe positioned between wells 1230 as shown in FIG. 16A to preventdiffusion between wells 1230 when liquid is being flowed into a well1230 via the upper flow channel 1264; and over a well 1230 as shown inFIG. 16B to contain a reaction in a well 1230 (or otherwise preventdiffusion between wells 1230) when liquid is being flowed into the well1230 via the lower flow channel 1262. Of course, droplet 1290 may alsoserve as a cap over a corresponding well 1230 even in cases where liquidis not being flowed into the well 1230 via the lower flow channel 1262.

In the example shown in FIGS. 16A-16B, a single droplet 1290 is sized tocover just one single well 1230. In other variations, the droplet 1290is larger and is sized to cover two or more wells 1230 simultaneously.Droplets 1290 may thus be utilized to selectively cover wells 1230 ingroups; rather than just covering single wells 1230 individually. Stillother suitable configurations and arrangements will be apparent to thoseskilled in the art in view of the teachings herein.

The electrode elements in the electrode layer 1284 may be selectivelyactivated by a controller that is directly integrated into the flow cell1200. By way of example only, such an integrated controller may beincorporated into a CMOS chip. In some such versions, the same CMOS chipor other controller also controls other features of the flow cell 1200(e.g., the valve 780, the electrode assembly 740, etc.). As anotherillustrative alternative, the electrode elements in the electrode layer1284 may be selectively activated by a controller that is directlyintegrated into a cartridge (e.g., the removable cartridge 200) thatreceives the flow cell 1200. As still another illustrative alternative,the electrode elements in the electrode layer 1284 may be selectivelyactivated by a controller that is directly integrated into the baseinstrument 102. Moreover, components of the controller that selectivelyactivates the electrode elements in the electrode layer 1284 may bedistributed among two or more of the flow cell 1200, a cartridge thatreceives the flow cell 1200, or the base instrument 102. Varioussuitable components and arrangements that may be utilized to providecontrol of the electrode elements in the electrode layer 1284 will beapparent to those skilled in the art in view of the teachings herein.

F. Electrode Arrangements to Prevent Diffusion

FIG. 17 shows another example of a flow cell 1300 that may be used toread and write polynucleotides as described herein. The flow cell 1300of this example includes an upper body portion 1302 and a lower bodyportion 1304. An upper fluid flow channel 1364 is defined between theupper and lower body portions 1302, 1304 and is operable to receive aflow of fluid (e.g., a fluid containing nucleotide bases, etc.). In thepresent example, the flow cell 1300 does not include a lower fluid flowchannel like other flow cells 600, 601, 700, 800, 900, 1000, 1100described herein. In some other versions, the flow cell 1300 doesinclude a lower fluid flow channel like other flow cells 600, 601, 700,800, 900, 1000, 1100 described herein.

The flow cell 1300 of this example further includes a plurality of wells1330 that are formed as recesses in the bottom surface 1312 of the upperfluid flow channel 1364. These wells 1330 are substantially similar tothe wells 630 described above. The flow cell 1300 further defines aplurality of interstitial spaces 1314 between the wells 1330. As notedabove, it may be desirable to minimize the size of these interstitialspaces 1314 to thereby maximize the density of wells 1330 within theflow cell 1300. In variations where the flow cell 1300 includes a lowerfluid flow channel, the bottom of each well 1330 may include an openingproviding a pathway for fluid communication between the well 1330 andthe lower fluid flow channel. Such an opening may include a valve thatis operable to selectively open or close to thereby selectively permitor prevent fluid communication from the lower fluid flow channel to thecorresponding well 1330.

An electrode assembly 1340 is positioned at the bottom of each well1330. The electrode assembly 1340 may be configured and operable justlike the electrode assembly 640 described above. In some versions, theelectrode assembly 1340 comprises an active copper element that isoperable to drive a redox reaction as part of the process formachine-writing DNA in the well 1330. The flow cell 1300 of this examplefurther includes electrode assemblies 1320 in the interstitial spaces1314 between the wells 1330, at the lower surface 1312 of the upperfluid flow channel 1364. During the writing process, the electrodeassembly 1340 and the electrode assemblies 1320 may be activatedsimultaneously such that the electrode assemblies 1320 provide generatea current that is reversed in comparison to the current generated by theelectrode assemblies 1340. This may effectively sharpen the boundariesof the electrode assembly 1340, which may in turn prevent diffusionbetween adjacent wells 1130.

As described above, light may be utilized to read machine-writtenpolynucleotides within the wells 1330. In some versions, one or moreexternal light sources is/are used to provide the light. In some otherversions, one or more internal light sources is/are used to provide thelight. In either case, the light emitted toward the wells 1330 mayultimately reach the polynucleotides within the wells 1330, which maycause fluorophores associated with those polynucleotides to fluoresce.The fluorescence emitted by the fluorophores associated with thepolynucleotides may be detected by image sensors 1392 in the lower bodyportion 1304 of the flow cell 1300. The image sensors 1392 are coupledwith a lower integrated circuit layer 1390 that is also positioned inthe lower body portion 1304 of the flow cell 1300. In the presentexample, the image sensors 1392 are in communication with the lowerintegrated circuit layer 1390 In this context, “in communication” meansthat the lower integrated circuit layer 1390 is in electricalcommunication with the image sensors 1392. For instance, the lowerintegrated circuit layer 1390 may be operable to receive and processsignals from the image sensors 1392, with the signals representingimages that are picked up by the image sensors 1392. “In communication”in this context may also include the lower integrated circuit layer 1390providing electrical power to the image sensors 1392.

By way of example only, the lower integrated circuit layer 1390 andimage sensors 1392 may be part of a CMOS chip. In some other variations,the lower integrated circuit layer 1390 and image sensors 1392 areintegrated into some other component. By way of example only, the lowerintegrated circuit layer 1390 and/or image sensors 1392 may beintegrated into a cartridge (e.g., the removable cartridge 200) thatreceives the flow cell 1300; may be integrated into the base instrument102; or may be integrated in some other component.

Each image sensor 1392 is positioned under a corresponding well 1330.Thus, when light source(s) is/are activated to emit light toward thewell(s) 1330, the corresponding image sensor(s) 1392 is/are configuredto detect fluorescence emitted by fluorophores associated withpolynucleotides (e.g., machine-written DNA) contained within the well(s)1330. The fluorescent light profile detected by image sensors 1392 maybe utilized to read the polynucleotides as described herein. As shown inFIG. 17, each electrode assembly 1340 defines an opening 1342 thatallows the fluorescence emitted by fluorophores associated withpolynucleotides (e.g., machine-written DNA) contained within the well(s)1330 to pass through the center of the electrode assembly 1340 tothereby reach the corresponding image sensor 1392. To define such anopening, each electrode assembly 1340 may have an annular shape or anyother suitable shape.

As is also shown in FIG. 17, the same integrated circuit layer 1390 maybe utilized to control the electrode assemblies 1320, 1340 and processimage signals captured by the image sensors 1392. In other words, theintegrated circuit layer 1390 may be in communication with the electrodeassemblies 1320, 1340. Of course, this configuration may be optional. Inother versions, different circuit layers or different circuit componentsmay be utilized to control the electrode assemblies 1320, 1340 and/orprocess image signals captured by the image sensors 1392.

VIII. Features to Prevent Optical Cross-Talk Between Adjacent Wells of aFlow Cell

In order to maximize the amount of data that may be stored in a DNAstorage device, it may be beneficial to maximize the number of wells 630in a flow cell 600, 601 of the DNA storage device. However, increasingthe well density in a flow cell 600, 601 may increase the risk ofoptical cross-talk between wells, particularly when the flow cellcomprises an optically transmissive material (e.g., glass, etc.). Suchoptical cross-talk may compromise the reliability of reading data from,or writing data to, the DNA storage device. It may therefore bedesirable to include features that prevent such optical cross-talk.Several illustrative examples of optical cross-talk prevention featuresare described in greater detail below. While the following examples areprovided separately with reference to separate drawings, it should beunderstood that the features of the following examples may be combinedin numerous ways in the same flow cell. Thus, the optical cross-talkprevention features described below should not be viewed as beingexclusive of each other

A. Optical Elements to Prevent Optical Cross-Talk

FIG. 18 shows an example of a flow cell 1700 that may be used to readand write polynucleotides as described herein. The flow cell 1700 ofthis example includes an upper body portion 1702, a middle body portion1704, and a lower body portion 1706. An upper fluid flow channel 1764 isdefined between the upper and middle body portions 1702, 1704 and isoperable to receive a flow of fluid (e.g., a fluid containing nucleotidebases, etc.). A lower fluid flow channel 1762 is defined between themiddle and lower body portions 1704, 1706 and is operable to receive aseparate flow of fluid (e.g., a fluid containing deblocking/deshieldingagents, etc.).

The flow cell 1700 of this example further includes a plurality of wells1730 that are formed as recesses in the bottom surface 1712 of the upperfluid flow channel 1764. These wells 1730 are substantially similar tothe wells 630 described above. The flow cell 1700 further defines aplurality of interstitial spaces 1714 between the wells 1730. As notedabove, it may be desirable to minimize the size of these interstitialspaces 1714 to thereby maximize the density of wells 1730 within theflow cell 1700. An electrode assembly 1740 is positioned at the bottomof each well 1730. The electrode assembly 1740 may be configured andoperable just like the electrode assembly 640 described above. Thebottom of each well 1730 includes an opening 1760 providing a pathwayfor fluid communication between the well 1730 and the lower fluid flowchannel 1762. In some versions, this opening 1760 includes a valve thatis operable to selectively open or close to thereby selectively permitor prevent fluid communication from the lower fluid flow channel 1762 tothe corresponding well 1730.

As described above, light may be utilized to read machine-writtenpolynucleotides within the wells 1730. To that end, FIG. 18 shows a setof light sources 1720 that are configured to emit light 1722 towardcorresponding wells 1730. While each well 1730 has a corresponding lightsource 1720 in this example, other versions may provide a single lightsource 1720 that is usable for a plurality of wells 1730. For instance,in such versions, the light source 1720 may be movable relative to theflow cell 1700 to selectively illuminate different wells 1730.Alternatively, the light source 1720 may be fixed in position and theflow cell 1700 may move relative to the light source 1720. In someversions, the light source(s) 1720 is/are external to the flow cell1700. For instance, the light source(s) 1720 may be integrated into acartridge (e.g., the removable cartridge 200) that receives the flowcell 1700; may be integrated into the base instrument 102; or may beintegrated in some other component. In some other versions the lightsource(s) 1720 is/are integrated directly into the flow cell 1700 (e.g.,within the upper body portion 1702).

The flow cell 1700 of the present example further includes an integratedcircuit layer 1790 with a plurality of image sensors 1792. By way ofexample only, the integrated circuit layer 1790 and image sensors 1792may be part of a CMOS chip. The integrated circuit layer 1790 and imagesensors 1792 are integrated into the lower body portion 1706 in thisexample, with the image sensors 1792 being positioned at the lowersurface 1766 of the lower fluid flow channel 1762. In some othervariations, the integrated circuit layer 1790 and image sensors 1792 areintegrated into some other component. By way of example only, theintegrated circuit layer 1790 and/or image sensors 1792 may beintegrated into a cartridge (e.g., the removable cartridge 200) thatreceives the flow cell 1700; may be integrated into the base instrument102; or may be integrated in some other component.

Each image sensor 1792 is positioned under a corresponding well 1730.Thus, when light source(s) 1720 is/are activated to emit light 1722toward the well(s) 1730, the corresponding image sensor(s) 1792 is/areconfigured to detect fluorescence emitted by fluorophores associatedwith polynucleotides (e.g., machine-written DNA) contained within thewell(s) 1730. The fluorescent light profile detected by image sensors1792 may be utilized to read the polynucleotides as described herein.

In order to ensure that light 1722 emitted by a light source 1720 onlyreaches the intended well 1730, the flow cell 1700 of the presentexample further includes an optical assembly 1780 within the upper bodyportion 1702 of the flow cell 1700. Each well 1730 has an associatedoptical assembly 1780 in this example. The optical assembly 1780 mayinclude one or more beam-shaping components. In some versions, theoptical assembly 1780 includes an optical waveguide, light pipe, oroptical fiber. In addition, or in the alternative, the optical assembly1780 may include a lens assembly that is configured to focus theintensity of the light 1722 on a lower region of the corresponding well1730.

In some other variations, the light sources 1720 are positionedunderneath the wells 1730, such as below or adjacent to the electrodeassembly 1740. Alternatively, the light sources may be positioned at oralong the sidewall of each well 1730. As another illustrative variation,the light sources 1720 may be positioned remotely relative to the wells1730, and the optical assemblies may communicate the light to the wells1730 via optical fibers, light pipes, or other light-conveyingstructures.

B. Light Sources to Prevent Optical Cross-Talk

FIG. 19 shows another example of a flow cell 1800 that may be used toread and write polynucleotides as described herein. The flow cell 1800of this example includes an upper body portion 1802, a middle bodyportion 1804, and a lower body portion 1806. An upper fluid flow channel1864 is defined between the upper and middle body portions 1802, 1804and is operable to receive a flow of fluid (e.g., a fluid containingnucleotide bases, etc.). A lower fluid flow channel 1862 is definedbetween the middle and lower body portions 1804, 1806 and is operable toreceive a separate flow of fluid (e.g., a fluid containingdeblocking/deshielding agents, etc.).

The flow cell 1800 of this example further includes a plurality of wells1830 that are formed as recesses in the bottom surface 1812 of the upperfluid flow channel 1864. These wells 1830 are substantially similar tothe wells 630 described above. The flow cell 1800 further defines aplurality of interstitial spaces 1814 between the wells 1830. As notedabove, it may be desirable to minimize the size of these interstitialspaces 1814 to thereby maximize the density of wells 1830 within theflow cell 1800. An electrode assembly 1840 1840 is positioned at thebottom of each well 1830. The electrode assembly 1840 1840 may beconfigured and operable just like the electrode assembly 640 describedabove. The bottom of each well 1830 includes an opening 1860 providing apathway for fluid communication between the well 1830 and the lowerfluid flow channel 1862. In some versions, this opening 1860 includes avalve that is operable to selectively open or close to therebyselectively permit or prevent fluid communication from the lower fluidflow channel 1862 to the corresponding well 1830.

As described above, light may be utilized to read machine-writtenpolynucleotides within the wells 1830. To that end, FIG. 19 shows a setof light sources 1822 that are configured to emit light towardcorresponding wells 1830. While each well 1830 has a corresponding lightsource 1822 in this example. The light sources 1822 are all coupled withan upper integrated circuit layer 1820 and are positioned to be flushwith the upper surface 1824 of the upper flow channel 1864. The upperintegrated circuit layer 1820 is operable to selectively drive the lightsources 1822 independently of each other. By way of example only, theupper integrated circuit layer 1820 may include a CMOS chip. By way offurther example only, the light sources 1822 may include microscopiclight emitting diodes (microLEDs). In some versions, each light source1822 for each well 1830 consists of a single microLED. In some otherversions, each light source 1822 for each well 1830 consists of an arrayof microLEDs. It should be understood that the use of microLEDs mayprovide greater precision in the delivery of light to selected wells1830, thereby minimizing the risk of the emitted light undesirablyreaching an adjacent well.

The flow cell 1800 of the present example further includes a lowerintegrated circuit layer 1890 with a plurality of image sensors 1892. Byway of example only, the lower integrated circuit layer 1890 and imagesensors 1892 may be part of a CMOS chip. The lower integrated circuitlayer 1890 and image sensors 1892 are integrated into the lower bodyportion 1806 in this example, with the image sensors 1892 beingpositioned at the lower surface 1866 of the lower fluid flow channel1862. In some other variations, the lower integrated circuit layer 1890and image sensors 1892 are integrated into some other component. By wayof example only, the lower integrated circuit layer 1890 and/or imagesensors 1892 may be integrated into a cartridge (e.g., the removablecartridge 200) that receives the flow cell 1800; may be integrated intothe base instrument 102; or may be integrated in some other component.

Each image sensor 1892 is positioned under a corresponding well 1830.Thus, when light source(s) 1822 is/are activated to emit light towardthe well(s) 1830, the corresponding image sensor(s) 1892 is/areconfigured to detect fluorescence emitted by fluorophores associatedwith polynucleotides (e.g., machine-written DNA) contained within thewell(s) 1830. The fluorescent light profile detected by image sensors1892 may be utilized to read the polynucleotides as described herein.

C. Polarizers to Prevent Optical Cross-Talk

FIG. 20 shows another example of a flow cell 1900 that may be used toread and write polynucleotides as described herein. The flow cell 1900of this example includes a first body portion 1904 and a second bodyportion 1906. An upper fluid flow channel 1964 is defined above thefirst body portion 1904 and is operable to receive a flow of fluid(e.g., a fluid containing nucleotide bases, etc.). In some versions, anupper body portion (e.g., like the upper body portion 1702, 1802described above) is positioned over the first body portion 904 tofurther define the upper fluid flow channel 1964. A lower fluid flowchannel 1962 is defined between the first and second body portions 1904,1906 and is operable to receive a separate flow of fluid (e.g., a fluidcontaining deblocking/deshielding agents, etc.).

The flow cell 1900 of this example further includes a plurality of wells1930 that are formed as recesses in the bottom surface 1912 of the upperfluid flow channel 1964. These wells 1930 are substantially similar tothe wells 630 described above. The flow cell 1900 further defines aplurality of interstitial spaces 1914 between the wells 1930. As notedabove, it may be desirable to minimize the size of these interstitialspaces 1914 to thereby maximize the density of wells 1930 within theflow cell 1900.

A light source 1920 is positioned above the wells 1930 and is configuredto emit light 1922 toward the wells 1930. In the example shown in FIG.20, the light source 1920 is configured to emit a beam of light 1922that may reach at least two wells 1930. In some versions, the lightsource 1920 is configured to emit a beam of light 1922 that may reachmany more than two wells 1930. In some other versions, each well 1930has its own dedicated light source 1920, such that the beam of light1922 from each light source 1920 is intended to only reach onecorresponding well 1930.

An electrode assembly 1940 is positioned at the bottom of each well1930. The electrode assembly 1940 may be configured and operable justlike the electrode assembly 640 described above. The bottom of each well1930 includes an opening 1960 providing a pathway for fluidcommunication between the well 1930 and the lower fluid flow channel1962. In some versions, this opening 1960 includes a valve that isoperable to selectively open or close to thereby selectively permit orprevent fluid communication from the lower fluid flow channel 1962 tothe corresponding well 1930.

As described above, light 1922 from the light source 1920 may beutilized to read machine-written polynucleotides within the wells 1930.The light 1922 emitted toward the wells 1930 may ultimately reach thepolynucleotides within the wells 1930, which may cause fluorophoresassociated with those polynucleotides to fluoresce. The fluorescenceemitted by these fluorophores may be detected by image sensors 1992 atthe bottom surface 1966 of the lower fluid flow channel 1962. The imagesensors 1992 are coupled with a lower integrated circuit layer 1990 thatis positioned in the second body portion 1906 of the flow cell 1900. Byway of example only, the lower integrated circuit layer 1990 and imagesensors 1992 may be part of a CMOS chip. In some other variations, thelower integrated circuit layer 1990 and image sensors 1992 areintegrated into some other component. By way of example only, the lowerintegrated circuit layer 1990 and/or image sensors 1992 may beintegrated into a cartridge (e.g., the removable cartridge 200) thatreceives the flow cell 1900; may be integrated into the base instrument102; or may be integrated in some other component.

Each image sensor 1992 is positioned under a corresponding well 1930.Thus, when light source(s) is/are activated to emit light toward thewell(s) 1930, the corresponding image sensor(s) 1992 is/are configuredto detect fluorescence emitted by fluorophores associated withmachine-written DNA contained within the well(s) 1930. The fluorescentlight profile detected by image sensors 1992 may be utilized to read thepolynucleotides as described herein.

When wells 1930 are densely packed together in a flow cell 1900, suchthat the interstitial spaces 1914 are very small, and one light source1950 is utilized to illuminate two or more adjacent wells 1930, it maybe difficult to selectively illuminate just one well 1930 withoutsimultaneously illuminating an adjacent well 1930. Similarly, it may bedifficult to illuminate two non-adjacent wells 1930 simultaneously withthe same light source 1950 without also simultaneously illuminating anywells 1930 that are adjacent to the targeted wells 1930. The selectiveillumination of particular wells 1930 with a single light source 1950that is capable of illuminating two or more wells 1930 may be desirablein cases where it is desired to only read polynucleotides that have beenwritten in the targeted wells 1930.

To provide enhanced control of illumination of wells 1930 with a singlelight source 1950 (or at least a light source 1950 that is operable toilluminate two or more wells 1930 simultaneously), the flow cell 1900 ofthe present example includes electrically activated polarizers 1920located in the upper regions 1936 of the wells 1930. Each polarizer 1920has a corresponding activation controller 1922 that is operable toselectively activate the polarizer 1920 to transition between apolarizing state and a non-polarizing state (e.g., by selectivelyapplying a voltage to the polarizer 1920 or removing the voltage fromthe polarizer 1920). When the polarizer 1920 is activated, the polarizermay provide an optical barrier between the light source 1950 and thewell 1930 associated with the polarizer 1920. When the polarizer 1920 isnot activated, the polarizer 1920 may allow light 1952 from the lightsource 1950 to reach the well 1930. Thus, when it is desired to allowthe light 1952 to reach some targeted wells 1930 without reaching other,non-targeted wells 1930, the polarizers 1920 of the targeted wells 1930may be left in a non-activated state (to thereby allow light 1952 topass through the polarizers 1920 of the targeted wells (1930)); whilethe polarizers 1920 of the non-targeted wells 1930 may be in anactivated state (to thereby prevent light 1952 from passing through thepolarizers 1920 of the non-targeted wells (1930)). In other words, thepolarizers 1920 may be utilized to prevent light 1952 from reachingwells 930 that are not intended to receive light 1952.

Also in the present example, each polarizer 1920 has a respectiveopening 1924 that is configured to allow fluid to flow from the upperfluid flow channel 1964 into the well 1930. While this opening 1924 issized to accommodate the flow of fluid therethrough, the opening 1924 isstill small enough to not meaningfully affect the ability of thepolarizer 1920 to effectively block light 1952 from the light source1950 when the polarizer 1920 is activated. In other words, even with theopening 1924, the polarizer 1920 may substantially permit fluidcommunication through the polarizer 1920 while substantially preventingoptical communication through the polarizer 1920. Some other versions ofthe polarizers 1920 may lack openings 1924. For instance, some suchversions may be utilized in variations of the flow cell 1900 that lackthe upper fluid flow channel 1964. Such variations of the flow cell 1900may provide fluid communication to the wells 1930 only via the lowerfluid flow channel 1962 (or through some other route). Some othervariations of the flow cell 1900, where the polarizers 1920 lackopenings 1924, may provide alternative paths for fluid flow from theupper fluid flow channel 1964 into the wells 1930. For instance, thefirst body portion 1904 may define additional flow paths for each well930, fluidically coupling the upper fluid flow channel 1964 with eachwell 1930 via the sidewall of the well 1930 (i.e., to a point under thepolarizer 1920).

D. Methods to Prevent Optical Cross-Talk or Otherwise Provide Efficiency

In addition to employing any of the various different hardwareconfigurations described above to prevent optical cross-talk betweenadjacent wells of a flow cell, various techniques may be utilized toprevent optical cross-talk between adjacent wells of a flow cell orotherwise provide efficiency in reading and/or writing machine-writtenDNA. Examples of such techniques are described in greater detail below.It should be understood that the below described techniques may beutilized in any of the flow cells 600, 601, 1700, 1800, 1900 describedherein or in any other suitable flow cell.

FIG. 21 shows an example of a method that employs a fragmenting approachto writing data, by showing a preference to leaving pixels or wellsadjacent to a written pixel or well empty. It should be understood thatdata may also be read at a faster speed it is known that the adjacentpixels or wells are empty; and the read speed may be reduced if it isknown that the adjacent pixels or wells have some contents. Leavingadjacent pixels or wells empty may also enable a more rapid transitionfrom a read-action on a pixel or well to a write-action on an adjacentpixel or well.

The process depicted in FIG. 21 begins with identifying 2200 unusedwells within a flow cell. For each unused well that is identified, theprocess includes determining 2202 whether the unused well is adjacent toa used well. If the identified unused well is in fact adjacent to a usedwell, then the process continues with evaluating 2204 the unused wellsto find a well that is not adjacent to a used well. When the processidentifies an unused well that is not adjacent to a used well, theprocess proceeds with placing 2206 that unused well in a first subset.The process then includes determining 2208 whether there are sufficientunused wells form a first subset. This determination may be based on theamount of data that needs to be stored and/or other factors. If thereare not yet enough unused wells in the first subset, then the processcontinues evaluating 2204 the unused wells to find another well that isnot adjacent to a used well. The above process may be repeated untilenough unused wells are placed in the first subset.

Once the first subset of unused wells is complete, the process proceedsto activating 2210 the electrode assemblies for those wells of the firstsubset to generate machine-written polynucleotides in those wells. Insome scenarios, the process ends at this stage. It should also beunderstood that it may be unnecessary to activate any barrier features,such as those described above to prevent cross-talk, before activating2210 the electrode assemblies to generate machine-writtenpolynucleotides in the wells of the first subset. In particular, sincethe adjacent wells do not contain any machine-written DNA at this stageof the process, there is no need for concern with cross-talk in thoseadjacent wells.

In some other scenarios, the process then proceeds to identifying 2212more unused wells. For each unused well, the process then determines2214 whether the unused well is adjacent to a used well. If theidentified unused well is not adjacent to a used well, then the processcontinues evaluating 2216 the unused wells to find a well that isadjacent to a used well. When the process identifies an unused well thatis adjacent to a used well, the process proceeds to placing 2218 thatunused well in a second subset. Then, a barrier feature associated withthat unused well in the second subset is activated 2220 beforeinitiating a write procedure in that unused well. With the barrierfeature activated, the electrode assembly for that well is thenactivated 2222 to generate machine-written polynucleotides in that well.In this example, only one unused well was placed in the second subset.In some other versions, more than one unused well may be placed in thesecond subset, such that the process may include a determination ofwhether the second subset is complete (before moving on to activating2220 one or more barrier features).

In some scenarios, a charge or “charge tag” may be added to a nucleotideor to an enzyme to reduce the power demand on electrode assemblies 640,1740, 1840 1840, 1940 during a writing process. Reducing the demand onelectrode assemblies 640, 1740, 1840 1840, 1940 during a writing processmay allow the form factor of electrode assemblies 640, 1740, 1840 1840,1940 to be reduced. Reducing the demand on electrode assemblies 640,1740, 1840 1840, 1940 during a writing process may also reduce theelectrical power load on the circuit and associated components thatdrive the electrode assemblies 640, 1740, 1840 1840, 1940. Adding chargetags to a nucleotide or to an enzyme may also allow for selective“recruitment” and “de-recruitment” via selective polarity of thenucleotides and/or enzymes to a given well 630, 1730, 1830, 1930. Suchrecruitment and de-recruitment may include concentrating the desirednucleotide or substrate-enzyme complex by electrostatic attraction(“recruitment”); or doing the reverse by repulsion (“de-recruitment”).

IX. Miscellaneous

All of the references, including patents, patent applications, andarticles, are explicitly incorporated by reference herein in theirentirety.

The foregoing description is provided to enable a person skilled in theart to practice the various configurations described herein. While thesubject technology has been particularly described with reference to thevarious figures and configurations, it should be understood that theseare for illustration purposes only and should not be taken as limitingthe scope of the subject technology.

As used herein, an element or step recited in the singular and proceededwith the word “a” or “an” should be understood as not excluding pluralof said elements or steps, unless such exclusion is explicitly stated.Furthermore, references to “one implementation” are not intended to beinterpreted as excluding the existence of additional implementationsthat also incorporate the recited features. Moreover, unless explicitlystated to the contrary, implementations “comprising” or “having” anelement or a plurality of elements having a particular property mayinclude additional elements whether or not they have that property.

The terms “substantially” and “about” used throughout this Specificationare used to describe and account for small fluctuations, such as due tovariations in processing. For example, they may refer to less than orequal to ±5%, such as less than or equal to ±2%, such as less than orequal to ±1%, such as less than or equal to ±0.5%, such as less than orequal to ±0.2%, such as less than or equal to ±0.1%, such as less thanor equal to ±0.05%.

There may be many other ways to implement the subject technology.Various functions and elements described herein may be partitioneddifferently from those shown without departing from the scope of thesubject technology. Various modifications to these implementations maybe readily apparent to those skilled in the art, and generic principlesdefined herein may be applied to other implementations. Thus, manychanges and modifications may be made to the subject technology, by onehaving ordinary skill in the art, without departing from the scope ofthe subject technology. For instance, different numbers of a givenmodule or unit may be employed, a different type or types of a givenmodule or unit may be employed, a given module or unit may be added, ora given module or unit may be omitted.

Underlined and/or italicized headings and subheadings are used forconvenience only, do not limit the subject technology, and are notreferred to in connection with the interpretation of the description ofthe subject technology. All structural and functional equivalents to theelements of the various implementations described throughout thisdisclosure that are known or later come to be known to those of ordinaryskill in the art are expressly incorporated herein by reference andintended to be encompassed by the subject technology. Moreover, nothingdisclosed herein is intended to be dedicated to the public regardless ofwhether such disclosure is explicitly recited in the above description.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein.

What is claimed is:
 1. An apparatus, comprising: (a) a body defining anupper flow channel a plurality of wells, the upper flow channel toreceive a flow of fluid containing nucleotides, each well of theplurality of wells being fluidically coupled with the corresponding flowchannel, each well of the plurality of wells having a floor with anaperture; (b) a lower channel positioned under the floor of each well ofthe plurality of wells, the lower channel to receive fluid containing adeblocking agent; (c) a plurality of valves, each valve of the pluralityof valves to transition between an open and closed state, each valve ofthe plurality of valves to permit fluid communication between the lowerchannel and the corresponding well of the plurality of wells in the openstate, each valve of the plurality of valves to reduce fluidcommunication between the lower channel and the corresponding well ofthe plurality of wells in the closed state; and (d) a plurality ofelectrodes, each electrode of the plurality of electrodes beingpositioned in a corresponding well of the plurality of wells, theplurality of electrodes to effect writing of polynucleotides in thecorresponding wells of the plurality of wells.
 2. The apparatus of claim1, further comprising an imaging assembly to capture images indicativeof one or more nucleotides in a polynucleotide.
 3. The apparatus ofclaim 1, each valve of the plurality of valves comprising a hydrogelmaterial.
 4. The apparatus of claim 1, each valve of the plurality ofvalves comprising an electroactive polymer.
 5. The apparatus of claim 1,each valve of the plurality of valves comprising a heat swellablepolymer.
 6. The apparatus of claim 1, each valve of the plurality ofvalves being biased toward a closed state, each valve of the pluralityof valves to open in response to fluid pressure against the valveexceeding a threshold.
 7. An apparatus, comprising: (a) a flow cell bodydefining one or more flow channels and a plurality of wells, each flowchannel of the one or more flow channels to receive a flow of fluid,each well of the plurality of wells being fluidically coupled with thecorresponding flow channel of the one or more flow channels, each wellof the plurality of wells defining a corresponding depth; (b) aplurality of electrodes, each electrode of the plurality of electrodesbeing positioned in a corresponding well of the plurality of wells, theplurality of electrodes to effect writing of polynucleotides in thecorresponding wells of the plurality of wells; and (c) a temperaturegradient generator, the temperature gradient generator to provide atemperature profile that varies between the plurality of wells.
 8. Theapparatus of claim 7, further comprising an imaging assembly to captureimages indicative of one or more nucleotides in a polynucleotide.
 9. Theapparatus of claim 7, the temperature gradient generator to provide arelatively higher temperature within each well of the plurality of wellsand a relatively lower temperature in spaces between the plurality ofwells.
 10. A method comprising: flowing fluid through a flow cell, thefluid containing nucleotides, the flow cell comprising a flow cell bodydefining one or more flow channels, each flow channel of the one or moreflow channels to receive a flow of fluid, at least one of the one ormore flow channels having: (i) a floor, (ii) a plurality of wellsdefined as recesses in the floor, each well of the plurality of wellsbeing fluidically coupled with the corresponding flow channel of the oneor more flow channels, the floor of the flow channel of the one or moreflow channels defining interstitial spaces between adjacent wells of theplurality of wells, and (iii) a plurality of barrier features above thewells of the plurality of wells or in the wells of the plurality ofwells; activating electrode assemblies at bottom regions of each well ofthe plurality of wells to generate machine-written polynucleotideswithin each well of the plurality of wells, the machine-writtenpolynucleotides representing stored data; and activating the pluralityof barrier features to reduce optical cross-talk between adjacent wellsof the plurality of wells.
 11. The method of claim 10, activating theelectrode assemblies at the bottom region of each well of the pluralityof wells comprising generating a positive current, activating theplurality of barrier features comprising generating a negative current.12. The method of claim 10, activating the plurality of barrier featurescomprising adding a charge tag to at least some of the nucleotides. 13.The method of claim 12, each charge tag being positively charged. 14.The method of claim 13, each charge tag imparting a net positive chargeto the corresponding nucleotide of the nucleotides.
 15. The method ofclaim 12, each charge tag being negatively charged.
 16. The method ofclaim 12, each charge tag being attached to one or more regions of thecorresponding nucleotide of the nucleotides selected from a ribose, aphosphate group, or a base.
 17. The method of claim 12, each charge tagbeing cleaved before a ligation event.
 18. The method of claim 12, eachcharge tag being cleaved after a ligation event.